Narrow Albino Leaf 1 Is Allelic to CHR729, Regulates Leaf Morphogenesis and Development by Affecting Auxin Metabolism in Rice

Plant Growth Regul (2017) 82:175–186 DOI 10.1007/s10725-017-0249-4

ORIGINAL PAPER

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

Jing Xu1,2 · Li Wang2 · Mengyu Zhou2 · Dali Zeng2 · Jiang Hu2 · Li Zhu2 · Deyong Ren2 · Guojun Dong2 · Zhenyu Gao2 · Longbiao Guo2 · Qian Qian2 · Wenzhong Zhang1 · Guangheng Zhang2

Received: 10 September 2016 / Accepted: 5 January 2017 / Published online: 2 February 2017 © Springer Science+Business Media Dordrecht 2017

Abstract The leaf is the main site of photosynthe- combined effect with a plurality of genes that work within sis and an important component of the ideotype in rice. a network regulating leaf shape Moreover, this gene was Its development and morphogenesis directly affects rice involved in the synthesis, transport, and signaling of auxin, yield. The rice mutant, narrow albino leaf 1 (naal1), was and thereby affecting the development of the root system, obtained from a rice mutant population generated from leaf vascular bundles, leaf morphogenesis, as well as the an EMS-induced indica variety Shuhui527 (SH527), and development of panicle traits. is mainly characterized by reduced plant height, narrow and albino leaves, a reduced number of crown roots, and Keywords Rice (Oryza sativa L.) · Leaf morphogenesis · an increased number of trichomes. Map-based cloning and Auxin metabolism · Pleiotropism 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 methyla- Introduction tion of H3K4 and H3K27. NAAL1 was expressed in a con- stitutive form in the rice root, stem, leaf sheath, and pani- Leaf morphology is part of the super-rice ideotype and its cle. Further functional studies showed that NAAL1 had a 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 Jing Xu, and Li Wang have contributed equally to this work. photosynthesis, photosynthetic efficiency, and dry matter accumulation. Leaf morphogenesis is often subject to the Electronic supplementary material The online version of this article (doi:10.1007/s10725-017-0249-4) contains supplementary coordinated regulation of genetic inheritance, hormonal material, which is available to authorized users. signaling, as well as environmental factors (Huang 2003). The structure of cuticle, number and structure of vascu- * Qian Qian lar bundles, development of abaxial sclerenchyma, number [email protected] of mesophyll cells, and size and number of vesicular cells * Wenzhong Zhang may lead to changes in the polarity of a leaf. The devel- [email protected] opment of the leaf polarity determines leaf length, leaf * Guangheng Zhang width, curling of the leaf tip, and other key indicators of [email protected] rice leaf morphogenesis. In recent years, scientists have 1 Rice Research Institute of Shenyang Agricultural University / studied the regulatory mechanism for leaf morphogenesis Key Laboratory of Northern Japonica Rice Genetics at the molecular level, isolating and cloning many of the and Breeding, Ministry of Education and Liaoning Province / regulatory genes related to this regulation; among these Key Laboratory of Northeast Rice Biology and Genetics and Breeding, Ministry of Agriculture, Shenyang 110866, genes, abaxially curled leaf 1 (ACL1) (Li et al. 2010), rice Liaoning, China outermost cell-specific (ROC5) (Zou et al. 2011), rolling 2 State Key Laboratory of Rice Biology, China National Rice leaf14 (RL14) (Fang et al. 2012), Zn-finger transcription Research Institute, Hangzhou 310006, Zhejiang, China factor (OsZHD1) (Xu et al. 2014), SEMI-ROLLED LEAF1

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(SRL1) (Xiang et al. 2012), shallot-like 2 (SLL2) (Zhang amino transferase playing a key role in auxin synthesis in et al. 2015), R2R3-MYB transcription factor (OsMYB103L) rice (Yoshikawa et al. 2014), and PIN1 acts as an auxin- (Yang et al. 2014), and rolled and erect leaf 1 (REL1) (Chen carrier (Xu et al. 2005), plays a major role in auxin-depend- et al. 2015) lead to the inward or outward rolling of leaves ent adventitious root development and tillering, and it is by regulating the number, size, and distribution of bulli- expressed in the vascular tissues and primordial root. form cells on the adaxial surface. SHALLOT-LIKE1 (SLL1) The narrow-albino leaf 1 (naal1) is a single recessive (Zhang et al. 2009) and semi-rolled leaf2 (SRL2) (Liu et al. gene mutant that was obtained from mutant population 2016) promote adaxial development of the abaxial leaf sur- generated from the ethyl methane sulfonate (EMS)-induced face and propel the inward rolling of the leaf by regulating mutagenesis of the rice indica variety, SH527. In contrast the differentiation of abaxial sclerenchyma. Curly flag leaf1 to the wild-type SH527, the mutant was characterized as (CFL1) (Wu et al. 2011) affects leaf rolling by regulating dwarfed plants mainly with narrow and albino upper leaves, the development of cuticle. ADAXIALIZED LEAF1 (ADL1) a reduced shoot-to-root ratio, an increase in the number (Hibara et al. 2009) affects the adaxial development of of trichomes. Map-based cloning results of the mutant epithelial cells and mesophyll tissues of the abaxial sur- identified the regulatory genenamed NARROW ALBINO face, which differentiates into alveolar cells and instigates LEAF 1 (NAAL1) that is allelic to the reported gene leaf rolling to the abaxial leaf surface. OsAGO7 (Shi et al. CHR729/OsCHR4 that encodes a CHR4/MI-2-like protein 2007) regulates leaf rolling by affecting the development of (Hu et al. 2012; Zhao et al. 2012; Ma et al. 2015). In this the adaxial/abaxial surface. The leaf width is directly asso- study, the function of NAAL1 was studied and the roles of ciated with the number of leaf veins. When the leaf is nar- NAAL1 in rice leaf morphogenesis, metabolic auxin regula- row, the number of large and small leaf veins is reduced. tion, and the rice yield formation were examined. 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. Materials and methods 2008), NARROW LEAF 1 (NAL1) (Qi et al. 2008; Zhang et al. 2014), NARROW AND ROLLED LEAF 1 (NRL1) (Hu Plant materials et al. 2010), NARROW LEAF 2/NARROW LEAF 3 nal2/3 (Cho et al. 2013) and RICE MINUTE-LIKE1 (RML1) The mutant naal1 was identified from a rice mutant popula- (Zheng et al. 2016), the first two regulated leaf width by tion generated by EMS-induced mutagenesis of the indica affecting the biosynthesis and polar transport of auxin variety SH527. The mutant naal1 was crossed with Nip- respectively, while the last three by influencing the number, ponbare, and 1178 mutant plants were identified for gene shape, and size of the vascular tissues simultaneously. mapping. All plants were cultivated in the experimental Leaf development could be regulated by several groups fields at the China National Rice Research Institute, either of genes, mainly including YABBY genes (Dai et al. 2007), in Hangzhou, Zhejiang or Lingshui, Hainan during the nor- auxin synthesis related YUCCA genes (Yamamoto et al. mal growing season. 2007), and AUXIN RESPONSE FACTOR (ARF) (Tiwari et al. 2003). These genes was predominantly expressed in Measurement of leaf traits and chlorophyll content the primordium differentiation period and seeding development (Toriba et al. 2007; Cheng et al. 2007). In rice, the The width and length of the upper three leaves of six plants YABBY family affects the growth of lateral organs. Notably, in both the wild-type SH527 and the mutant naal1 were OsYAB3 (Dai et al. 2007), OsYAB4 (Liu et al. 2007) and measured at the heading stage and averaged for data analy- DROOPING LEAF (DL) (Nagasawa et al. 2003; Ohmori sis. Root traits were investigated using roots analysis sys- et al. 2011) are associated with leaf development in rice, of tem (WSEEN LA-S Company, China) in both the wild type which OsYAB4 is important for the vascular tissues devel- and the mutant. Other important agronomic traits, includ- opment and DL for the development of the leaf midrib, ing plant height, panicle length, primary branch number sheath, and floral organs. Zhao et al. (2001) has shown that per panicle, secondary branch number per panicle, number YUCCA is rate limiting in auxin synthesis in plants, thereby of grains per panicle, seed setting rate, 1000-grain weight, playing an important role in the biosynthetic pathway of grain length, and grain width, were measured according the tryptophan-dependent IAA. The expression of its fam- to the Standard Evaluation System for Rice (http://www. ily member OsYUCCA1 is limited to the tip of leaves, roots, knowledgebank.irri.org/ses/). and vascular tissues (Yamamoto et al. 2007). ARF regulates Fresh leaves of three independent plants were sampled the response to auxin and the development of the abaxial from the wild type SH527 and the mutant naal1 at the till- leaf surface (Waller et al. 2002; Wang et al. 2007). In addi- ering stage, and were collected and chlorophyll content was tion, the rice gene FISH BONE (FIB) encodes a tryptophan measured. The leaf veins were excised and leaves were cut

1 3 Plant Growth Regul (2017) 82:175–186 177 into small pieces. Approximately 0.20 g of leaf tissue was for primary mapping by using simple sequence repeats placed into 25 ml of extraction solution containing 95% (SSR) markers. For the target region, six sequence-tagged acetone and absolute ethanol (2:1, v/v). Extraction sam- site (STS) markers were designed from the differences ples were incubated in the dark for 24 h at 28 °C, the opti- sequences between Oryza sativa L. ssp. japonica cultivar cal density (OD) at 663, 645, and 470 nm were measured Nipponbare and indica cultivar 93 –11 obtained from the using a DU800 spectrophotometer (Beckman Coulter, Ger- National Center for Biotechnology Information (http:// many). The methods used for pretreatment and chlorophyll www.ncbi.nlm.nih.gov/). The molecular markers used are determination were as previously described by Yang et al. listed in Table S1. (2016). Construction of vectors and plant transformation Scanning electron microscopy For functional complementation, a vector was constructed Leaves were taken from naal1 and wild-type SH527 plants by placing a NAAL1 full-length cDNA sequence driven by at the heading stage and placed in 2.5% glutaraldehyde for its native promoter that was amplified from the wild type more than 4 h, and rinsed three times with phosphate buffer plant. This construct was introduced into the mutant naal1 and fixed in 1%OsO 4 in phosphate buffer at 4 °C. Samples by Agrobacterium tumefaciens-mediated transformation. were washed in phosphate buffer for 15 min each time, then The method for the vector construction was described by dehydrated through gradient ethanol solutions each step for Zhao et al. (2012). The primers for the complementation 15 min. After that, samples were incubated in an ethanol- vector are listed in Table S1. isoamyl (1:1) acetate mixture for 30 min and transferred To determine the subcellular localization of NAAL1, the to isoamyl acetate for 1 h. Dried the samples with liquid coding sequence of NAAL1 without the stop codon was CO2, and coated the samples with gold–palladium, finally amplified and then inserted into the pCAMBIA1300-eGFP examined using a Tabletop Microscope (Hitachi TM-1000, vector to generate the 35S:NAAL1:GFP fusion construct. Japan). We then transformed the protein fusion construct and control vector into rice leaf protoplasts. The GFP signal was Transmission electron microscopy detected using an OLYMPUS IX71 confocal microscope. The GFP vector primers are listed in Table S1. Flag leaves taken from the mutant naal1 and wild type SH527 plants at the heading stage were placed in 2.5% RNA extraction and expression analysis glutaraldehyde and were placed under a vacuum until the leaves sank. Leaf samples were treated according to the To examine the expression pattern of NAAL1, we extracted method described by Zhao et al. (2015) and analyzed with a RNA of leaves, roots, stems, sheaths, and panicles from the transmission electron microscope (Hitachi H-7650, Japan). wild type SH527 using Axyprep Multisources Total RNA Miniprep Kit (USA-based Axygen Scientific, Inc). The Histology and microscopy RNA from leaves in both SH527 and naal1 were extracted from plants treated with 1-naphthaleneacetic acid (NAA). The flag leaf samples were collected from the mutant naal1 The RNA was reverse transcribed into cDNA using Rever- and the wild type SH527 at the heading stage, and fixed the Tra Ace qPCR RT Master Mix with gDNA Remover Kit samples in 50% FAA overnight at 4 °C. After that, the sam- (TOYOBO, JAPAN). The amplification program for SYBR ples were dehydrated using an ethanol gradient, infiltrated Green (95 °C, 10s; 60 °C, 30s; 72 °C, 15s, for 40 cycles) with dimethylbenzene, and embedded in paraffin and then was performed on an Applied Biosystems 7900 HT Fast sectioned into 10–12 μm slices, stained with 1% safranine Real-Time PCR System (USA) following the manufactur- and 1% Fast Green, dehydrated using ethanol and infil- er’s instructions. Three replicates from each cDNA sample trated with dimethylbenzene again. Finally, a microscope were used for Real-Time PCR. Each sample was normal- (NIKON ECLIPSE 90I, Japan) was used for examination ized to the amount of Actin transcript detected in the same of the cellular microstructure in the leaf samples (Ren et al. sample. The RT-PCR primers used are listed in Table S1. 2015). NAA treatment Map‑based cloning Seeds of the mutant naal1 and the wildtype SH527 were An F2 population of 4913 plants was derived from a cross germinated in a 1/2 × MS liquid culture medium and were between the mutant naal1 and Nipponbare plants. A total subjected to an NAA gradient treatment with six different of 1178 plants with mutant phenotypes were selected concentrations of exogenous auxin (0 nm, 0.1 nm, 1 nm,

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10 nm, 0.1 µm, and 1 µm). The phenotypes were observed upper-surface (adaxial) albino leaves and normal lower- after 7 days of growth. The shoot length, maximum root surface color (Fig. 1d). Beginning at the seedling stage, length, and the root number of three independent plants naal1 had a narrower and shorter leaf compared to that of in different concentrations of exogenous auxin were meas- wild-type SH527 (Fig. 1a, b, c). The phenotype observa- ured. The total root length was measured using the WSEEN tions and statistics showed that the average width of the LA-S Plant Roots Analysis System software. The relative upper three leaves of wild-type SH527 at the heading stage expression of NAAL1 in the mutant naal1 and wild type were 1.81, 1.61, and 1.48 cm, respectively. However, those SH527 seedlings grown in different NAA treatments was of naal1 were 0.63, 0.62, and 0.52 cm, which indicated an analyzed. The root samples of three independent plants that appreciable reduction of 65.2, 61.5, and 65.5%, respec- were grown in the free NAA treatment cultures were col- tively (Fig. 1g). Additionally, at the heading stage, the aver- lected at 7 days after sowing and used for examination of age length of the upper three leaves of the naal1 mutant the endogenous IAA content at the Institute of Botany, Chi- were significantly reduced by 12.15, 16.27, and 19.00 cm, nese Academy of Sciences (Beijing, China). respectively, when compared to SH527 (Fig. 1f). Another notable feature of the mutant was abnormal root growth, which was observed as a significant reduction in the num- Results ber of crown roots and total root length (Fig. 1e). At the seedling stage 7 days after sowing, the number of crown Phenotypic characterization of naal1 roots in the wild type SH527 and naal1 were 8.63 and 4.50, respectively, and their total root lengths were 56.06 In this study, the narrow-leaf albino mutant, naal1, was and 35.22 cm, respectively, for which naal1 relative to the successfully separated from a rice mutant population gen- wild-type SH527 dropped by 47.86 and 37.17%, respec- erated by EMS-induction of the indica variety SH527. tively (Fig. 1i, j).This results suggest that the reduction of The mutant was characterized as a dwarfed plant with the total root lengths of naal1 might be derived from the

a c SH527 g SH527 b f naal1 naal1

(cm) idth (cm)

Leaf w Leaf length

QG UG SH527 naal 1 SH527 naal1 )/ / / )/ QG/ UG/

d e h j i

r (cm)

(cm)

height Root numbe Plant Root length

SH527 naal1 SH527 naal1 SH527naal1 SH527 naal1 SH527QDDO

Fig. 1 Phenotypic characterization of naal1. a Cross-sections difference between WT (left) and naal1 (right). Bar 2 cm. f, g Com- through the middle of the mature flag leaves of wild-type (WT, parison of the upper three leaves between WT and naal1; leaf length Shuhui527, left) and naal1 (right) plants, bar 1 cm. b Morphological (f) and leaf width (g) at maturity. h Comparison of the plant height difference between WT (left) and naal1 (right) at the tillering stage, of WT and naal1 at maturity. i–j Comparison of the upper three bar 20 cm. c Comparison of the flag leaf of WT (left) and naal1 leaves between WT and naal1 in crown root number (i), and total root (right), bar 5 cm. d Differences of leaf shape and leaf color in the length (j) in 10-day old seedlings. *P < 0.05, **P < 0.01; significance adaxial-side between WT (left) and naal1 (right), bar 1 cm. e Root based on t-test

1 3 Plant Growth Regul (2017) 82:175–186 179 decrease of the number of crown roots. At the mature stage, membranes on the naal1 adaxial surface as compared to the plant height of naal1 was only 70.9% of that in SH527 SH527 (Fig. 2e, f, i, j). However, less apparent differences (Fig. 1h). Compared to SH527, the mature panicle traits were detected on the abaxial leaf surface (Fig. 2g, h, k, l) of naal1, including panicle length, number of secondary which data suggests that the upper-albino leaf phenotype in branches per panicle, the number of spikelets per panicle, naal1 is caused by changes in the chloroplast structure on seed setting rate, and 1000-grain weight, dropped sharply; the adaxial surface of the leaf. the number of primary branches per panicle and the grain To understand the narrow leaf phenotype in naal1, the length also markedly declined. However, no significant dif- numbers of main and secondary leaf veins were analyzed ference was found in the grain width (Table 1). NAAL1 not in wild-type SH527 and the mutant naal1 under a stereomi- only affects leaf morphology and root system development, croscope and by the paraffin method. As observed under but also has regulatory roles in panicle traits and the devel- the stereomicroscope, the number of the main and second- opment of some grain morphologies. ary veins in naal1 was reduced by 27.1 and 63.2%, respectively, compared to SH527 (Fig. 2q). The leaf transections NAAL1 affects the development of leaf trichomes, made from paraffin embedded leaves showed that the num- the vascular bundle, and chloroplasts ber of vascular bundles in naal1 leaves were reduced by approximately 20% compared to SH527, and the number of To observe changes in the leaf surface of the mutant, the mesophyll cells in the direct vicinity of the adaxial vesic- leaves of wild-type SH527 and naal1 mutant plants were ular cells was also significantly decreased in the mutant analyzed by scanning electron microcopy. Our observations (Fig. 2m, n). showed that the number of large and small trichomes of the adaxial and abaxial surfaces of naal1 leaves increased rela- Map‑based cloning of NAAL1 tive to wild type. The number of large and small trichomes of the adaxial surface was 2.96 and 3.68 times greater than To locate the regulatory genes related to leaf narrowing that of the wild type, respectively (Fig. 2 a, c, p) whereas in the mutant, naal1 was straightbred and reciprocally those on the abaxial surface was 2.58 and 1.98 times crossed with the japonica rice variety Nipponbare. The greater, respectively (Fig. 2b, d, o). F1-generation phenotype was identified and the separa- To examine the causes of the albino leaf phenotype in tion percentage of the F2-generation plants was based naal1, the chlorophyll content and chloroplast structure of on differences in leaf shape. A Chi square test results wild-type SH527 and mutant naal1 leaves at the peak of showed that a single recessive gene pair controls the the tillering stage were measured and observed by acetone mutant naal1 phenotype (P = 0.0978). A total of 1178 tissue processing and transmission electron microscopy individual mutants with the narrow-leaf phenotype were (TEM). The contents of chlorophyll a and b, and carot- chosen from the F2 population that consisted of 4913 enoids in SH527 at the tillering stage were 3.01, 0.94, and individual plants. Using genome-wide screening, the 0.55, respectively; while those in the naal1 mutant were NAAL1 gene was located in the region flanked by the 2.21, 0.68, and 0.45, respectively. This indicated a signifi- RM6835 and RM11 SSR markers on chromosome 7. cant reduction of key photosynthetic molecules by 26.6, Through further development of new markers and the use 27.7, and 18.2%, respectively, in the naal1 mutant (Fig. 2r). of map-based cloning, the gene was eventually targeted Additionally, the chloroplast structure in naal1 and between the STS markers P5 and P6 on BAC P0005E02 SH527 was observed by TEM showed fewer chloroplast at approximately 47.1 kb (Fig. 3a). The Rice Genome

Table 1 The panicle traits of Trait SH527 naal1 SH527 and naal1 rice plants Panicle length (cm) 26.63 ± 0.11 20.93 ± 0.61** Number of primary branches per panicle 12.67 ± 1.53 9.67 ± 0.0.58* Number of secondary branches per panicle 21.33 ± 0.58 9.33 ± 1.15** Number of spikelets per panicle 156.33 ± 10.26 105.67 ± 7.37** Seed setting rate (%) 84.26 ± 4.89 17.27 ± 8.46** Grain length (mm) 11.20 ± 0.41 10.52 ± 0.26** Grain width (mm) 2.67 ± 0.06 2.65 ± 0.08 1000-Grain weight (g) 36.78 25.32**

*, **Represents a significant difference between wild type and mutant at the 0.05 level and 0.01 level based on the t-test

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a b c d

200μm 200μm 200μm 200μm

e f g h

i j k l

0.2µm 0.2µm 0.2µm 0.2µm

m o p q SH527 SH527 SH527 SH527 r SH527 naal1 naal1 naal1 4 naal1

g/g ) 3 100μm (m

of adaxial side ** of abaxial side er ber scular bundles 2

n content naal 1 va num ll nu mb s s 1 ** me me ** Number of Chlorophy 100μm Tricho 0 Tricho Lg Trich Sm Trich Lg Trich Sm Trich LVB SVB chla chlbcar

Fig. 2 The differences in leaf trichome number, the vascular bundles, Cross-sections of flag leavesfrom WT m( ) and naal1 (n). o–p Com- and chloroplasts between wild type SH527 and the mutant naal1. parison of the number of trichomes on the abaxial side (o), and the a–b A scanning electron micrograph of the adaxial (a) and abaxial adaxial side (p) of WT and naal1 leaves (Lg Trich, large trichomes; (b) surfaces of leaves from WT. c–d A scanning electron micrograph Sm Trich, small trichomes). q Comparison of the number of vascu- of the adaxial (c) and abaxial (d) surfaces of leaves from naal1. lar bundles between WT and naal1 (LVB, the large vascular bundle; e–f Transmission electron micrographs of the adaxial surfaces of SVB, the small vascular bundle). r Comparison of the chlorophyll leaves from WT (e) and naal1 (f). g–h Transmission electron micro- content between WT and naal1 (chla, chlorophyll a; chlb, chlorophyll graphs of the abaxial surfaces of leaves from WT (g) and naal1 (h). b; car, carotenoid). *P < 0.05, **P < 0.01; significance based on the i–j Larger images of E and F. k–l Larger images of G and H. m–n t-test (WT, Shuhui527)

Annotation Databases (http://rice.plantbiology.msu.edu/ The complementation vector that contained the wild- cgi-bin/gbrowse/rice/) predicted that four possible open type gene LOC_Os07g31450 sequence and its native pro- reading frames (ORFs) were present within the interval moter fragment was constructed. Through agrobacterium- that encodes an unknown protein, a CHR4/MI-2-like pro- mediated transformation, the mutant was transformed and tein, a peptide N-asparagine amidase, or a MYB family the functional complementation was verified by the iden- transcription factor, respectively. Sequencing and com- tification of the transgene. Nine independent lines were parison with the wild-type SH527 indicated that LOC_ obtained from the transgenic progeny, which exhibited nor- Os07g31450 encodes the CHR4/MI-2-like protein, and mal leaf morphology. Among these lines, two independent that this gene was mutated on the 6052nd base, creat- transgenic lines were grown in paddy field and observed for ing a substitution from an A to a T in the mutant naal1 complementation of the naal1 mutant phenotypes (Fig. 3c). (Fig. 3b). The LOC_Os07g31450 is thought to be NAAL1 The leaf phenotype indicated that the leaf narrowing, that controls the leaf width. upper-albino leaf, and other mutant defects characteristic of

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RM11 a Markers RM6835 P1 P3 P5P6 P4 P2 (GFP) and driven by 35S promoter. It was then transformed into rice leaf protoplasts. Using an Olympus IX71 confocal CHR.7 microscope, the expression of the 35S:NAAL1:GFP con- OJ1457_D07 OJ1197_D06 struct was observed in the nucleus, which is indicative of a BACs P0005E02 nucleoprotein (Fig. 4b).

Altered transcription level of auxin‑ and leaf naal1 Recombinants(38) (3) (2)(1)(0) (3) (8) (38) development‑related genes in

NAAL1 Leaf development is subject to the coordinated control of Candidate Region genes, for example, leaf development YABBY genes, auxin 18619454 18666555 synthesis related genes such as YUCCA genes, auxin trans- 6052 port-associated genes, and other leaf development related Gene 0 12895 b genes. To better understand the regulatory mechanism of ATG A T TAG NAAL1 that controls leaf morphogenesis, the expression of c d genes related to leaf development was compared in young developing leaves of wild-type SH527 and mutant naal1 plants. The qRT-PCR results showed that compared with the wild-type SH527, the expression of YAB1, YAB4, YAB7, YUC1, YUC2, YUC4, YUC7, YUC8, ARF2, ARF5, and PINB1 was significantly downregulated in naal1, whereas that of PIN1B (Wang et al. 2009) was markedly upregu- naal1-com Shuhui527 naal1 naal1-com-1, -2 lated (Fig. 5a). Moreover, the relative expression of SLL1, NAL7, NRL1, and NAL1, which are cloned genes related to Fig. 3 Map-based cloning of NAAL1. a Fine mapping of NAAL1 on rice leaf narrowing, were also significantly downregulated NAAL1 chromosome 7. was mapped primarily to the region flanked in the mutant; compared to the wild type, the expression by RM6835 and RM11 on chromosome 7, and then narrowed to NAL1 naal1 a 47.101-kb region between P5 and P6 on BAC P0005E02. b Gene of was decreased to 3.6% in the mutant , and structure of NAAL1. A base substitution from A to T was sequenced NRL1 was declined to 54.1% in naal1 leaves (Fig. 5c). in the fifth exon of the ORF of thenaal1 mutant. c Phenotypes of These results show that the narrow-leaf phenotype of naal1 two independent complementation lines in transgenic rice plants at may be related to the altered transcriptional activity of leaf the heading stage grown in the field.d Flag-leaf morphology of WT, naal1, naal1-complementation-line-1 (naal-com-1), and naal1-com- development-related and auxin-related genes. plementation-line-2 (naal-com-2) Response to different exogenous NAA concentrations on naal1 development the naal1 mutation were complemented in these transgenic plants (Fig. 3d). This complementation test showed that To verify that NAAL1 was associated with the auxin met- LOC_Os07g31450 is the target gene NAAL1, which regu- abolic pathway, the expression of FIB (Yoshikawa et al. lates leaf width. NAAL1 is also allelic to CHR729/OsCHR4 2014), a key gene for the auxin tryptophan metabolic path- (Hu et al. 2012; Zhao et al. 2012; Ma et al. 2015), which way, was analyzed. Results showed that the expression of regulates the methylation of H3K4 and H3K27. NAAL1 in the mutant naal1 was decreased to 12.5% of that in wild type, thereby indicating that naal1 mutant plants Expression pattern and subcellular localization had defects in auxin synthesis in which resulted in plant of the NAAL1 protein growth and morphological abnormalities (Fig. 5a). Thus, we examined endogenous IAA content of total plants in To characterize the expression pattern of NAAL1, quanti- SH527 and naal1 at the seedling stage and found signifi- tative real-time PCR (qRT-PCR) was applied to determine cant differences (Fig. 5b). Content of endogenous IAA in the expression of NAAL1 in different tissues and organs of naal1 was declined by 12.5% compared with SH527. the wild-type SH527. Results showed that NAAL1 was con- To test this hypothesis, the seeds of both wild-type stitutively expressed in the rice root, stem, leaf sheath, and SH527 and the mutant naal1 were sown on 1/2 × MS cul- panicle; the expression in the root was the highest, whereas ture medium and subjected to a NAA gradient treatment that in the panicle was the lowest (Fig. 4a). Meanwhile, to upon germination. The NAA treatments consisted of six identify the specific localization of NAAL1 in the cell, the different concentrations of exogenous auxin (0 nm, 0.1 nm, ORF of NAAL1, was fused to the green fluorescent protein 1 nm, 10 nm, 0.1 µm, and 1 µm NAA) and the plant

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Fig. 4 Expression pattern of a NAAL1 and the subcellular 1.6 NAAL1 localization of the pro- 1.4 tein in WT. a Relative expression levels of NAAL1 in leaf, OsNAAL1 1.2 of root, stem, sheath, and panicle. 1.0 b Subcellular localization of the 0.8 NAAL1 protein in normal rice ssion level leaf protoplasts e 0.6 pr

ex 0.4 0.2

Relative 0 leaf root stem sheath panicle

b 35S::GFP

10um

35S::OsNAAL1::GFP

10um

phenotypes were observed after 7 days of growth (Fig. 6a, measured in plants grown in different NAA concentrations b). Results showed that without exogenous NAA, the shoot were consistent with the variation in NAAL1 expression. length of SH527 was similar to that of naal1, the total root When considering the wild type SH527 phenotype and the length (including lateral roots) was 8.12 cm longer than expression of NAAL1, combined with the response to exog- that of naal1, and the maximum root length was 2.44 cm enous auxin (NAA), the results showed that the metabolic shorter than that of naal1. With increasing NAA con- equilibrium of endogenous auxin was disrupted and the centrations, the shoot length, maximum root length, and normal expression of NAAL1 was inhibited, thereby affect- total root length of SH527 decreased gradually. However, ing normal plant growth and development. By contrast, in 1 nm NAA, the shoot length and total root length of naal1 the naal1mutant, the mutation in NAAL1 affected the auxin increased by 19.7 and 41.9%, respectively, compared to metabolic pathway and caused a decline in the synthesis those conditions free NAA (Fig. 6c, f). When the concen- of endogenous auxin. With the addition of 1 nm NAA, the tration of NAA increased to 1 µm, the growth of the roots expression reached maximum, and the root system devel- and shoots of SH527 and naal1 were inhibited to the great- opment and plant growth of the mutant naal1 was partly est extent (Fig. 6c–f). recovered. Thus, the NAAL1 gene is involved in the tryp- Without exogenous NAA, the expression of NAAL1 in tophan biosynthesis pathway of auxin production, thereby SH527 was twice that in the naal1 mutant (Fig. 6g). With affecting root development and leaf morphogenesis. 0.1 nm NAA, the expression of NAAL1 in SH527 was rapidly reduced by 64.6% to only one-third of the normal level. With increasing concentrations of exogenous NAA, Discussion the expression always remained 20–40% that of the normal level in wild type. Under all NAA conditions, the expres- In this study, we identified a narrow albino leaf mutant, sion of NAAL1 in the naal1 mutant was higher than that and used map-based cloning to found the underlying gene of the wild-type SH527. In naal1, the expression in plants and named NAAL1 which was allelic to reported gene treated with 1 nm NAA was 1.87 times that of plants not CHR729/OsCHR4. NAAL1 affected the development treated with NAA. When the concentration of exogenous and morphogenesis of leaf and root by involving in the NAA increased to 1 µm, no significant difference was synthesis, transport, and signaling of auxin. In addition, observed in the wild type. The shoot and root lengths the effects of NAAL1 on the development of chloroplast

1 3 Plant Growth Regul (2017) 82:175–186 183

a 1.8 1.6 * 1.4 1.2 1.0 0.8 * * ** * 0.6 ** ** ** * ** ** ** ** Relative expression level 0.4 ** 0.2 ** 0 FIB WT ARF 5 YAB3 ARF 1 ARF 2 ARF 3 ARF 4 YUC6 YUC7 YUC8 YUC9 YAB1 YAB2 YAB4 YAB5 YAB6 YUC1 YUC2 YUC3 YUC4 YUC5 PIN1 PIN2 PIN3 <$% PIN1B PIN1C NAAL1

b 0.06 c 1.2 SH527 naal1 l

/g ) 0.05 1 µg ( 0.04 * 0.8 * 0.03 0.6 * 0.02 0.4

Content of IAA 0.01 0.2 **

Relative expression leve ** 0 0 SH527 naal1 SLL1 NAL7 NRL1 NAL1

Fig. 5 The mutant naal1 has altered expression levels of auxin- and els of NAAL1 and auxin-related genes (FIB, YUC family, ARF family, leaf development-related genes, and content of endogenous IAA. The PIN family and YAB family) in naal1. b Content of endogenous IAA rice Actin gene was used as an internal positive control to normal- in SH527 and naal1. c Transcription levels of four cloned narrow leaf ize the expression level of each gene investigated. Total RNA was genes in naal1 were normalized to their levels in WT. Each column extracted from leaves in the seedling stage. a Quantitative real time represents the mean ± SD of three biological replicates. *P < 0.05, PCR (qRT-PCR) analysis was performed to study the transcript lev- **P < 0.01; significance based on t-test

and trichomes were also observed. The effects of NAAL1 details on the specific molecular mechanism are still function on gene expression important to plant mor- pending. phogenesis varied at different gene loci. For example, Leaf development is also regulated by a variety of Hu et al. (2012) found that CHR729, which is a CHD3 phytohormones, especially at the stage of leaf primor- protein with dual capabilities in regulating chromatin, dium differentiation, and the metabolism of auxin greatly not only recognizes and modulates the methylation of affects leaf morphogenesis (Bar and Ori 2015). Among H3K4 and H3K27 that can inhibit tissue-specific genes, the isolated regulatory genes responsible for leaf shape, but it is also involved in the morphogenesis of multi- many genes are associated with the auxin metabolic path- ple organs in plants. Zhao et al. (2012) has shown that way, for example, NAL1, a regulatory gene that controls CHR729/OsCHR4 could play an important role in the the polar transport of auxin, affects leaf width (Qi et al. development of chloroplast in the mesophyll cells on the 2008; Zhang et al. 2014). NAL7, a member of the YUCCA adaxial leaf surface, and the mutation of this gene could gene family, is involved in the biosynthesis of auxin and induce albino features on the adaxial surface. Ma et al. also regulates leaf width (Fujino et al. 2008). In the pre- (2015) determined that CHR729 regulates seedling devel- sent study, we show that the expression of some members opment via the gibberellin biosynthetic pathway. There- of YUC (a gene family in the tryptophan-dependent bio- fore, NAAL1 may be involved in multiple regulatory synthetic pathways), ARF (auxin transporters), and PIN pathways and play important roles in the morphogenesis (auxin carriers) families change significantly in naal1 of major tissues and organs including roots, leaves, and when compared to wild type. Moreover, the expression panicles, thus showing pleiotropic effects in rice. Differ- of FIB, a key gene related to the biosynthetic pathway ent genetic networks could regulate the effects of NAAL1 of auxin (tryptophan), was decreased to 12.5% in naal1 on the development of different tissues and organs; and when compared to the wild type. As for root development,

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a b SH527 2cm naal1 2cm

0M 0.1nM 1nM 10nM 0.1uM1uM 0M 0.1nM1nM 10nM 0.1uM1uM

SH527 SH527 c 16.0 naal1 d naal1 14.0 m) 12.0 m) 10.0 8.0 6.0 4.0 Shoot length (c 2.0 Max root length (c 0 00.1nM 1nM 10nM0.1uM 1uM 00.1nM 1nM 10nM0.1uM 1uM f SH527 80.0 SH527 14.0 e naal1 m) 70.0 naal1 12.0 60.0 be r 10.0 50.0 8.0 40.0 6.0 30.0

Root num 4.0 20.0

2.0 Total root length (c 10.0 0 0.0 00.1nM 1nM 10nM 0.1uM 1uM 0 0.1nM 1nM 10nM0.1uM

1.2 SH527 g naal1 1.0 NAAL1

0.8

0.6

0.4

0.2

Relative expression 0 00.1nM 1nM 10nM0.1uM 1uM

Fig. 6 Response to exogenous NAA concentration in naal1. a–b obtained from three biological replicates. g Relative mRNA levels of Phenotypes of 7-day-old wild type (WT, Shuhui527) and naal1 NAAL1 in leaves from 7-day-old seedlings of WT and naal1 treated plants treated with or without an NAA gradient. c–f Effects of exog- with NAA. Mean and SD values were obtained from three biological enous NAA treatment on shoot length (c), maximum root length (d), replicates root number (e), and total root length (f). Mean and SD values were the role the NAAL1 mutation played was also observed. mutant and the wild type were shown (Fig. 6). Similar The max root length in free NAA culture was longer in results were found in mutant fib for rice root development naal1 than in wild type. When application of NAA, low (Yoshikawa et al. 2014), which played a pivotal role in concentrations of NAA (an exogenous auxin), partially IAA biosynthesis in rice. In addition, in naal1 plants, the restored the root growth of the mutant and the expression expression of some genes related to transport and signal- of NAAL1 was the highest, while with the increasing of ing of auxin had significant differences with the wild type concentrations, the inhibition effects of NAA on both the SH527 (Fig. 5). Thus, we speculate that NAAL1 takes

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