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RESEARCH ARTICLE 1
2
NONSTOP GLUMES 1 Encodes a C2H2 Zinc Finger Protein that 3
Regulates Spikelet Development in Rice 4
5
Hui Zhuanga,1, Hong-Lei Wanga,1, Ting Zhanga,1, Xiao-Qin Zenga, Huan Chena, 6
Zhong-Wei Wanga, Jun Zhanga, Hao Zhenga, Jun Tanga, Ying-Hua Linga, Zheng-7
Lin Yanga, Guang-Hua Hea,2 and Yun-Feng Lia,2 8
9 aRice Research Institute, Key Laboratory of Application and Safety Control of 10
Genetically Modified Crops, Academy of Agricultural Sciences, Southwest University, 11
Chongqing 400715, China 12
13 1These authors contributed equally to this work. 14 2Corresponding authors: 15
Yun-Feng Li, email: [email protected], Telephone number: +8615923964839; 16
Guang-Hua He, email: [email protected]; Telephone number: 86-023-68250158. 17
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Short title: NSG1 regulates spikelet development in rice 19
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One sentence summary: NSG1, encoding a member of the C2H2 zinc finger protein 21
family, plays a pivotal role in maintaining organ identities in the spikelet by repressing 22
the expression of LHS1, DL, MFO1. 23
24
The authors responsible for distribution of materials integral to the findings presented in 25
this article in accordance with the policy described in the Instructions for Authors 26
(www.plantcell.org) are: Yunfeng Li ([email protected]) and Guang-Hua He 27
([email protected]) 28
29
ABSTRACT 30
The spikelet is an inflorescence structure unique to grasses. The molecular mechanisms 31
underlying spikelet development and evolution are unclear. In this study, we 32
characterized three allelic recessive mutants in rice (Oryza sativa), nonstop glumes 1-1 33
(nsg1-1), nsg1-2, and nsg1-3. In these mutants, organs including such as the rudimentary 34
glume, sterile lemma, palea, lodicule, and filament were elongated and/or widened, or 35
transformed into lemma- and/or marginal region of the palea (mrp)-like organs. NSG1 36
encoded a member of the C2H2 zinc finger protein family and was expressed mainly in 37
the organ primordia of the spikelet. In the nsg1-1 mutant spikelet, LHS1, DL and MFO1 38
were ectopically expressed in two or more organs, including the rudimentary glume, 39
sterile lemma, palea, lodicule, and stamen, whereas G1 was down-regulated in the 40
rudimentary glume and sterile lemma. Furthermore, the NSG1 protein was able to bind to 41
regulatory regions of LHS1, and then recruit the co-repressor TPRs to repress expression 42
by down-regulating histone acetylation levels of the chromatin. The results suggest that 43
NSG1 plays a pivotal role in maintaining organ identities in the spikelet by repressing the 44
expression of LHS1, DL, and MFO1.45
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Plant Cell Advance Publication. Published on December 5, 2019, doi:10.1105/tpc.19.00682
©2019 American Society of Plant Biologists. All Rights Reserved
2
INTRODUCTION 47
Inflorescence development has a major effect on crop yield, and involves three types of 48
transformation: The transition of the shoot apical meristem (SAM) from vegetative to 49
reproductive development causes a change of the branching pattern that leads to the 50
generation of a more complex structure, the inflorescence meristem (IM), which then 51
produces several primary-branch meristems (PBMs) until termination. Each PBM 52
continues to generate next-order meristems laterally until they acquire floral meristem 53
(FM) identity. Floral organs develop from the flanks of the FMs, ultimately yielding 54
seeds (Ikeda et al., 2004). The rice (Oryza sativa) panicle includes three types of IMs, 55
namely primary branch meristems (PBMs), secondary branch meristems (SBMs), and 56
spikelet meristems (SMs) (Schmidt and Ambrose, 1998; McSteen et al., 2000). The 57
grain number per panicle in rice (which is almost equal to the number of SMs) is 58
determined mainly by the number of PBMs and SBMs. The majority of high-yield genes 59
reported recently are involved in regulation of panicle branching, including GRAIN 60
NUMBER 1A (Gn1a), Ghd7, DENSE AND ERECT PANICLE 1 (DEP1), DENSE AND 61
ERECT PANICLE 2 (DEP2), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 62
14/IDEAL PLANT ARCHITECTURE 1 (OsSPL14/IPA1) and TAWAWA 1 (TWA1), which 63
have made important contributions to increases rice yield (Ashikari et al., 2005; Xue et 64
al., 2008; Huang et al., 2009; Li et al., 2010a; Miura et al., 2010; Jiao et al., 2010; 65
Yoshida et al., 2013). Note that the spikelet is also an inflorescence unit and can produce 66
one or more florets in some species of grass, which offers the possibility of increasing the 67
number of florets per spikelet to improve yield in species that nowadays produce only a 68
one-floret/grain spikelet, such as rice. However, to achieve this, an enhanced 69
understanding of the genetic and molecular mechanism of spikelet development is 70
required. 71
In rice, the spikelet contains one fertile terminal floret (consisting of four whorls of 72
floral organs: one lemma and one palea, two lodicules, six stamens, and one pistil), one 73
pair of sterile lemmas, and one pair of rudimentary glumes. Although wild-type rice 74
produces just a single grain per spikelet, the loss of determinacy of SMs may enable an 75
increase in the number of florets and/or other organs per spikelet, as demonstrated from 76
studies of mutants of genes such as SUPERNUMERARY BRACT GENE (SNB), 77
3
INDETERMINATE SPIKELET 1 (IDS1), and MULTI-FLORETS SPIKELET 1 (MFS1) 78
(Lee and An, 2012; Ren et al., 2013). In these mutants, the transition from SM to FM is 79
delayed, which leads to the production of an additional floret(s). 80
An additional possible means of increasing the number of florets per spikelet is by 81
restoring the two “lateral florets”, which now are considered to be antecedents of “sterile 82
lemmas”. Two prevailing hypotheses for the origin and identity of sterile lemmas (as well 83
as rudimentary glumes) have been proposed. One hypothesis holds that sterile lemmas 84
and rudimentary glumes are reduced glumes (Schmidt and Ambrose, 1998). The second 85
hypothesis suggests that the spikelet of rice ancestors contained a terminal floret and two 86
lateral florets. According to the latter proposal, the rice spikelet is a “three-florets 87
spikelet” and the lateral florets degenerated into sterile lemmas during evolution (Arber, 88
1935; Kellogg, 2009; Yoshida et al., 2009; Kobayashi et al., 2010). Compared with the 89
ample morphological evidence in support of the first hypothesis, abundant genetic and 90
molecular evidence has accumulated in support of the second hypothesis from research 91
on genes such as LONG STERILE LEMMAS (G1), EXTRA GLUME 1 (EG1), PANICLE 92
PHYTOMER 2 (PAP2), ABERRANT SPIKELET AND PANICLE 1 (ASP1), LEAFY HULL 93
STERILE 1 (LHS1), and LATERAL FLORET 1 (LF1) (Li et al., 2009a; Yoshida et al., 94
2009; Yoshida et al., 2012; Wang et al., 2013; Lin et al., 2014; Wang et al.,2017; Zhang et 95
al., 2017). The loss of function of G1, EG1, PAP2, and ASP1, and overexpression of 96
LHS1 lead to transformation of sterile lemmas into lemma-like organs, whereas the gain 97
of function of LF1 results in development of a lateral floret (including the palea, lodicule, 98
stamens, and pistil) in the axil of the sterile lemma. Therefore, it is possible to gain 99
additional, complete (fertile and morphologically normal) lateral florets by combining the 100
two types of mutants (Zhang et al., 2017). Nevertheless, some molecular evidence 101
supports the first hypothesis. In the mfs1 mutant, the sterile lemma is degenerated and 102
assumes the identity of a rudimentary glume, which implies that the sterile lemma is 103
homologous to the rudimentary glume (Ren et al., 2013). Therefore, additional research is 104
needed to gain an improved understanding of this issue. 105
In the previous study, we identified preliminary a spikelet mutant non-stop glumes 106
(nsg1-1) and mapped the NSG1 gene to a region on chromosome 4 (Wang et al., 2013). In 107
present study, we characterized the other two allelic recessive mutants of NSG1, namely 108
4
nsg1-2 and nsg1-3. In the three allelic mutants, the rudimentary glume, sterile lemma, 109
palea, lodicule, and filament were elongated and/or widened, and were transformed into 110
lemma and/or mrp-like organs. Our data further indicate that NSG1 encodes a C2H2 111
transcription repressor and plays a pivotal role in the regulation of lateral organ identities 112
in the spikelet by repression of the expression of floral organ identity genes LHS1, 113
DROOPING LEAF (DL), and MOSAIC FLORAL ORGANS 1 (MFO1). 114
115
116
RESULTS 117
Three allelic recessive mutants of NSG1, designated nsg1-1, nsg1-2, and nsg1-3, were 118
identified in the current study. Given that nsg1-1 showed more severe defects than the 119
other two mutants, the remainder of this study focuses on the nsg1-1 mutant. 120
121
Defective Organ Identity in the nsg1-1 Spikelet 122
The wild-type (WT) rice spikelet is composed of one pair of rudimentary glumes, one 123
pair of sterile lemmas (also termed empty glumes), and one terminal fertile floret. The 124
floret consists of two additional bracts, the lemma and palea, which encase three whorls 125
of floral organs: two lodicules, six stamens and a central carpel (Horshikawa, 1989) 126
(Figure 1A). The rudimentary glumes, sterile lemmas, and lemma/palea are considered to 127
be three pairs of glume-like organs, inserted on the rachilla with distichous phyllotaxy 128
from bottom to top. The sizes of the lemma and palea are much larger than the 129
rudimentary glumes and sterile lemmas, and the lemma is wider than the palea (Figures 130
1A1 and 1A8). Compared with the lemma, the palea consists of two parts—the main 131
body of the palea (bp) and two marginal regions of the palea (mrp) (Figures 1A3, 1A7, 132
and 1A8). The bp shows similar features to those of the lemma, with a silicified abaxial 133
epidermis bearing trichomes and protrusions (Figures 1A6 and 1A8). However, the mrp 134
structure is distinct in showing a smooth and non-silicified abaxial epidermis due to a 135
lack of trichomes and protrusions (Figures 1A7 and 1A8). The sterile lemma has a 136
smooth abaxial surface with a few trichomes and is larger than the rudimentary glume 137
(Figures 1A3 and 1A5). The two rudimentary glumes are much reduced, and abundant 138
trichomes and protrusions are observed on the abaxial surface (Figures 1A3 and 1A4). 139
5
In the nsg1-1 spikelets, the rudimentary glume and sterile lemma were elongated 140
and/or widened to various degrees. The inner rudimentary glume was elongated to the 141
same length as the sterile lemma in approximately 93% of nsg1-1 spikelets (Figures 1B4, 142
1D4, and 1G), of which approximately 1% showed a lemma-like epidermis with similar 143
protrusions (Figure 1D4). The outer and inner sterile lemmas were elongated and 144
widened in the majority of nsg1-1 spikelets, in which approximately 11% and 51%, 145
respectively, showed lemma-like features (Figures 1B–1E and 1G). In contrast to the 146
smooth epidermis of the WT sterile lemma, the nsg1-1 lemma-like sterile lemmas 147
displayed a rough abaxial epidermis similar to that of the WT lemma, in which the 148
tubercles and trichomes showed an orderly parallel arrangement (Figures 1B5, 1B6, 1D4–149
1D6, 1E4–1E6, and 1G). 150
In addition, a number of floral organs gained a lemma-like identity in the nsg1-1 151
spikelets. The mrps were transformed into a lemma-like structure in approximately 53% 152
of nsg1-1 spikelets. These lemma-like mrps were significantly wider than those of the 153
WT and showed a crooked structure resembling WT lemma. Histological analysis also 154
showed a silicified abaxial epidermis of these mrps instead of a smooth and non-silicified 155
one. Notably, these features of the abnormal mrps were characteristic of WT lemma 156
(Figures 1B7, 1B8, 1C6, and 1G). The lodicules were also elongated and widened, and/or 157
gained lemma and/or mrp-like identities in over one-third of nsg1-1 spikelets (Figures 158
1C4, 1B8, 1C6, and 1G). We also observed that the filaments of the stamens were 159
broadened and were transformed into lodicule/mrp-like organs in approximately 6% of 160
nsg1-1 spikelets (Figures 1C5, 1B8, 1C6, and 1G). 161
162
Histological and qPCR Analysis of Lateral Organs in nsg1-1 Spikelets 163
In accordance with these observations, almost all of the lateral organs (rudimentary 164
glumes, sterile lemmas, paleas, lodicules, and stamens except for the lemmas) tended to 165
be transformed into lemma-like organs, and the organs that were much nearer to the 166
position of the lemmas were more similar to the lemmas (Figure 1F). We therefore 167
further explored the histologic structure of these organs (Figure 2; Supplemental Figure 168
1). In a transverse section of WT lemma and bp, a number of vascular bundles and a four-169
6
layer structure which consisted of a silicified abaxial epidermis, several layers of fibrous 170
sclerenchyma cells, several layers of spongy parenchyma cells, and a vacuolated abaxial 171
epidermis were observed (Figures 1A8, 2A4 ; Supplemental Figure 1B). 172
Compared with the WT lemma and bp, the WT mrp consisted of a non-silicified 173
abaxial epidermis, a large number of spongy parenchyma cells, several fibrous 174
sclerenchyma cells, and a non-vacuolated abaxial epidermis (Figures 1A8, 2A4; 175
Supplemental Figure 1C). With regard to the WT rudimentary glume, three tissues were 176
observed, namely an abaxial epidermis, an adaxial epidermis, and several layers of 177
spongy parenchyma cells, and there was no obvious vascular bundle (Figure 2A1). In the 178
WT sterile lemma, a single vascular bundle and three tissues similar to rudimentary 179
glumes were observed (Figures 1A8, 2A2–2A3; Supplemental Figure 1A). The WT 180
lodicule consisted of many parenchymatous cells and several interspersed tracheal 181
elements (Figure 2A5). The WT filament showed radial organization and consisted of one 182
central vascular bundle and several layers of parenchymatous cells (Figure 2A5). 183
In almost all elongated and/or widened nsg1-1 rudimentary glumes and sterile lemmas, 184
the number of vascular bundles was increased to 3–5, and several layers of ectopic 185
fibrous sclerenchyma cells were observed between the abaxial epidermis and parenchyma 186
cell layers (Figures 2B1–2B3, 2C1–2C3, 2D1–2D3; Figures S1D-1F). In addition, the 187
silicified abaxial epidermis and vacuolated adaxial epidermis specific to the WT lemma 188
and bp were observed in some of the elongated and/or widened nsg1-1 sterile lemmas but 189
not the rudimentary glumes (Figures 2C1, 2C3, 2D2, 2D3; Supplemental Figure 1F). 190
Some inner sterile lemmas showed a characteristic of the WT lemma with same cell 191
layers and number of vascular bundles (Figure 2D3). Therefore, the histologic 192
characteristics of nsg1-1 rudimentary glumes were very similar to the WT mrp, while 193
nsg1-1 sterile lemmas displayed the combined cell characteristics of the WT lemma and 194
mrp. In some nsg1-1 mrps, lodicules and filaments, ectopic cell layers of fibrous 195
sclerenchyma cells were formed to various degrees (Figures 2B4, 2B5, 2C4, 2C5, 2D4, 196
and 2D5). A silicified abaxial epidermis and vacuolated abaxial epidermis were observed 197
in several nsg1-1 mrps (Figure 2D4). These results indicated that nsg1-1 mrps gained a 198
7
partially lemma-like character, and nsg1-1 lodicules and filaments gained a partially mrp-199
like character. Taking together, these observations demonstrated that all of the lateral 200
organs in nsg1-1 spikelets, other than the lemma, had gained a lemma- and/or mrp-like 201
anatomical structure by differentiation of ectopic fibrous sclerenchyma cell layers, a 202
silicified abaxial epidermis and/or vacuolated abaxial epidermis. In addition, we 203
concluded that an organ gained a stronger lemma-like identity with increased proximity 204
to the lemma. 205
Next, the mRNA levels of certain organ identity/marker genes were detected to further 206
determine the identity of these defective organs, namely DL (mainly expressed in the 207
lemma and pistil and specifies their identity) (Yamaguchi et al., 2004), LHS1 (mainly 208
expressed in the lemma and bp, and specifies their identity) (Wang et al., 2010), and 209
MFO1 (mainly expressed in the mrp and lodicule and specifies their identity) (Li et al., 210
2011). In nsg1-1 inner rudimentary glumes, MFO1 was ectopically expressed, which 211
suggested that these organs gained a mrp-like identity (Figure 2E). In nsg1-1 outer sterile 212
lemmas, LHS1, DL and MFO1 were ectopically expressed, which suggests that these 213
organs gained a partial lemma- and/or mrp- identity (Figures 2E–2G). In nsg1-1 inner 214
sterile lemmas, MFO1 expression was not detected, whereas LHS1 and DL were 215
ectopically expressed at an increased level, which indicated that these organs gained a 216
stronger lemma identity (Figures 2E–2G). In nsg1-1 paleas, up-regulation of DL and 217
down-regulation of MFO1 expression were observed, which demonstrates that these 218
organs gained a lemma-like identity (Figures 2E–2G). In nsg1-1 lodicules, up-regulation 219
of DL expression was detected, which similarly implies that a lemma-like identity was 220
gained (Figure 2F). In nsg1-1 stamens, DL and MFO1 expression levels were increased, 221
suggesting that these organs gained a lemma-, mrp- and lo-like identity (Figures 2F and 222
2G). Thus, the expression patterns of these genes were consistent with the phenotypic 223
observations. 224
225
Similarity of Defects in the nsg1-2 and nsg1-3 Mutants to Those of the nsg1-1 226
Mutant 227
The nsg1-2 and nsg1-3 mutants showed very similar defects in morphology, histology, 228
8
and expression patterns of genes associated with organ identity compared with those of 229
the nsg1-1 mutant (Figures S2 and S3). However, some differences were observed. Outer 230
rudimentary glumes were absent in almost all nsg1-1 spikelets examined (Figures 1B–1E 231
and 1G) and were absent in approximately 78% of nsg1-3 spikelets (Figures S2B and 232
S3B). By contrast, this phenotype was not observed in nsg1-2 spikelets, thus a pair of 233
rudimentary glumes were retained in the nsg1-2 spikelets (Figures S2A and S3A). In 234
addition, no obvious defects were observed in the outer sterile lemma of nsg1-2 spikelets, 235
whereas more than 90% of the outer sterile lemmas were transformed into a lemma-like 236
organ in nsg1-1 and nsg1-3 spikelets (Figure 1G; Figures S3A and S3B). The palea and 237
lodicule of nsg1-3 spikelets showed defects in a few cases, whereas the 238
mutation frequency was much higher in the nsg1-2 and nsg1-1 spikelets (Figure 1G; 239
Figures S3A and S3B). Taken together, these observations demonstrate that the nsg1-1 240
mutant showed more severe defects than the nsg1-2 and nsg1-3 mutants. 241
242
The nsg1-1 Mutant Exhibited Abnormal Early Spikelet Development 243
Young spikelets of both the WT plant and the nsg1-1 mutant were examined at 244
different developmental stages using scanning electron microscopy (Figure 3). The WT 245
sterile lemma primordia formed with alternate phyllotaxis during the spikelet 4 (Sp4) 246
stage (Figure 3A1) and developed quickly until the Sp8 stage (Figures 3A2–3A4), but 247
developed slowly after that. The nsg1-1 sterile lemma primordia were first visible at Sp4, 248
and thereafter differentiated and proliferated more rapidly than those of the WT during all 249
stages examined in this study (Figures 3B and 3C). The differentiation and proliferation 250
of WT rudimentary glume primordia were severely restricted, and no obvious change in 251
shape and size was detected from Sp4 to Sp8, compared with other organs in the spikelet, 252
such as the sterile lemma, lemma, and palea (Figure 3A). In nsg1-1 spikelets, however, 253
the inner rudimentary glume primordia may overcome the developmental restriction and 254
develop similarly to the sterile lemma and the lemma (Figures 3B and 3C). These 255
observations indicate that the transformation of a sterile lemma and rudimentary glume 256
into a lemma-like or lemma/sterile lemma-like organ might occur at an early 257
developmental stage in nsg1-1 spikelets. The absence of outer rudimentary glumes in 258
nsg1-1 spikelets was confirmed by detailed observation of spikelets at early 259
9
developmental stages (Figures 3B and 3C). 260
The WT primordia of lemma and palea formed successively during Sp4. From Sp4 to 261
Sp8, the primordia developed rapidly and finally hooked together. It was clear that the 262
size of palea primordia was always smaller than that of lemma primordia (Figure 3A). In 263
some nsg1-1 spikelets, however, the palea primordia was larger than that of WT, with the 264
same size of lemma primordia. The growth process and appearance of these palea 265
primordia were similar to lemma primordia as well suggesting that the palea primordia 266
had been transformed into lemma-like primordia (Figures 3B1, 3B2, 3C3, and 3C4). 267
Because it was difficult to distinguish the additional lemma from lemma-like organs 268
during the stages of primordia formation, only several additional lemmas were observed 269
with certainty when the normal lemma and palea primordia were visible in nsg1-1 270
spikelets (Figure 3C1). Some defects in floral organ primordia were observed in the nsg1-271
1 mutant. During the Sp5–Sp7 stages, several lodicule primordia were elongated and 272
widened, and visible in nsg1-1 spikelets (Figures 3B2 and 3C2), but none were visible in 273
WT spikelets (Figure 3A2). Thus, the abnormal lodicule primordia might have gained a 274
lemma-like identity. During the Sp5–Sp7 stages, the stamen primordia were spherical in 275
the WT (Figures 3A2 and 3A3), whereas the shape of some stamen primordia was 276
compressed in the nsg1-1 mutant (Figure 3C3). The abnormal stamen primordia might 277
have gained a lodicule-like or lemma-like identity. Taken together, all the defects 278
observed in mature spikelets were observed during stages of early spikelet development 279
in nsg1-1 spikelets. 280
281
Identification of NSG1 282
In a previous study, we fine-mapped NSG1 to a region on chromosome 4, and a 13 bp 283
insertion was identified locating after +444bp/147 amino acid (AA) of the Os04g36650 284
CDS in the nsg1-1 mutant (Wang et al., 2013). On the basis of previously published 285
sequence data, however, the chromosomal structure in the mapping region differs 286
between two cultivars, japonica rice ‘Nipponbare’ and indica rice ‘9311’, for which 287
whole-genome sequences are available. Between the two closest markers, the interval is 288
about 90 Kb in 9311 but only 15 Kb in Nipponbare (BAC: OSJNBa0058G03), because of 289
a large insertion located upstream of the Os04g36650 gene promoter (Wang et al., 2013). 290
10
Transformation of the Os04g36650 wild-type genomic fragment from Nipponbare BAC 291
OSJNBa0058G03 into the nsg1-1 mutant failed to complement the nsg1-1 defects, 292
although the transformation was repeated three times during 2011 to 2014. 293
In 2014, we identified a T-DNA insertion line at the Os04g36650 locus, RMD_ITL-294
04Z11DF46, from the Rice Mutant Database (RMD) (Zhang et al., 2007), which has a 295
background of the japonica cultivar ‘ZH11’ (Figure 4A). In this insertion line, the T-296
DNA insertion occurred in the -1202bp of Os04g36650 promoter, therefore expression of 297
the gene is almost undetectable (Supplemental Figure 4). Next, allelic verification 298
between nsg1-1 and the insertion line was undertaken. The nsg1-1 mutant, the insertion 299
line, and the F1 progeny displayed very similar phenotypes, which suggested that all were 300
allelic mutants of Os04g36650 (Figure 4C). Thus, the insertion line was designated as 301
nsg1-2. 302
We observed that the promoter sequence of nsg1-2 was located on Nipponbare BAC 303
OSJNBb0056O04 (but not on BAC OSJNBa0058G03). The BAC OSJNBb0056O04 had 304
been mapped to chromosome 4 in 2015 according to the genome information on the 305
website (http://signal.salk.edu/cgi-bin/RiceGE/), and its sequence was identical to that of 306
9311. The complementary vector was reconstructed using DNA of the WT cultivar ‘J10’ 307
as the PCR template and primers were designed in accordance with the sequence of BAC 308
OSJNBb0056O04. The WT genomic fragment of Os04g36650, which contained the 309
2,925 bp upstream sequence from the start codon and 525 bp coding sequence, was 310
cloned into the vector pCAMBIA1301 fused with the green fluorescent protein (GFP) 311
gene, and then transformed into the nsg1-1 mutant. The mutant phenotypes were rescued 312
to various degrees in different transgenic lines, some of which were completely restored 313
to the WT phenotype, such as No. 7 line. Other lines continued to develop elongated 314
rudimentary glumes, such as No. 1 and No. 3 lines (Figure 4B). We also identified an 315
additional allelic mutant, nsg1-3, in which a single-nucleotide substitution from A to T in 316
the +124bp of Os04g36650 CDS led an AA change from Arg to Trp and displayed 317
extremely similar phenotypes to those of the nsg1-1 and nsg1-2 mutants (Figure 4A; 318
Supplemental Figure 2A). Taken together, these results demonstrated that Os04g36650 319
was identical to NSG1. 320
We considered whether more than one copy of Os04g36650 was present in the genome 321
11
of Nipponbare, or the copy number differed between indica and japonica rice, on account 322
of the two BACs OSJNBa0058G03 and OSJNBb0056O04 of Nipponbare. DNA gel blot 323
analysis was used to analyze the genomic constitution of four rice cultivars, namely J10 324
(indica rice cultivar, the WT of nsg1-1), XD1B (indica rice cultivar, the WT of nsg1-3), 325
ZH11 (japonica rice cultivar, the WT of nsg1-2), and Nipponbare (japonica rice cultivar). 326
Using the promoter sequence of NSG1, which was unique to BAC OSJNBb0056O04, as 327
the probe, only one copy was detected among the four cultivars (Supplemental Figure 5A 328
and S5B). Similarly, probing with the coding sequence of NSG1, which was present on 329
the two BACs, also only one copy was detected (Supplemental Figure 5A and S5C). 330
These results indicate that Os04g36650 was a single-copy gene in both indica and 331
japonica rice, and was located in the region corresponding to BAC OSJNBb0056O04 on 332
chromosome 4. 333
334
NSG1 Encodes a C2H2 Zinc Finger Protein 335
NSG1 encodes a protein of 175 amino acids and shows high similarity to transcription 336
factors that contain a QALGGH-type single C2H2 zinc finger DNA-binding domain 337
(Figure 5A; Supplemental Figure 6 and S7B). This type of zinc finger domain has been 338
identified in several proteins involved in flower development, such as SUPERMAN 339
(SUP), JAGGED (JAG), and NUBBIN (NUB) in Arabidopsis thaliana and STAMENLESS 340
1 (SL1) in rice (Dinneny et al., 2004; Ohno et al., 2004; Dinneny et al., 2006; Xiao et al., 341
2009). 342
Phylogenetic analysis revealed that QALGGH-type single C2H2 zinc finger genes 343
from angiosperms formed a ‘NSG1-like’ clade, which was further resolved into three 344
subclades. Subclade 3 comprised all proteins from dicots, whereas the proteins from 345
monocots were placed in subclades 1 and 2. NSG1, its paralog Os02g35460, and several 346
orthologs from grasses were placed in subclade 1, whereas the paralog Os09g26210 and 347
several orthologs from grasses and other monocots were in subclade 2 (Figure 5A, 348
Supplemental datasets 1-3). 349
The protein motif prediction program MEME was used to analyze the full-length 350
amino acid sequences of NSG1, partial NSG-like proteins, SUP, JAG, and SL1. NSG1 351
shared very similar motif structures (motifs 1, 2, 3, and 6) with other proteins in subclade 352
12
1, although it lacked motifs 4 and 5. Almost all NSG1-like genes shared three extremely 353
similar motifs (motifs 1, 2, and 3), except At1g80730 with only Motif 1 and Motif 3. In 354
addition, SUP and JAG shared Motifs 1 and 2 with NSG1-like proteins, whereas SL1 355
shared only Motif 1 with NSG1-like proteins (Supplemental Figure 7A). Motif 1 was 356
characterized as a classic QALGGH-type C2H2 zinc finger DNA binding domain (Li et 357
al., 2013), while both motifs 2 and 3, located respectively at N- and C-termini, were 358
considered as the LxLxL-type ethylene-responsive element binding factor-associated 359
amphiphilic repression (EAR) motifs, which contain two distinct types (LxLxL and 360
DLNxxP) and are believed to specifically interact with corepressor TOPLESS(TPL)/TPL 361
RELATED PROTEIN (TPR) to restrict transcription of target genes (Ke et al., 2015). In 362
the nsg1-1 mutant, the 13bp insertion would lead to a frame shift after the first 147AA, 363
destroying the C-terminal EAR motif, while in the nsg1-3 mutant the AA change 364
occurred in the conserved C2H2 zinc finger DNA binding domain (Figure 4A, 365
Supplemental Figure 6). 366
To determine the subcellular localization of NSG1, the fluorescence signal of the 367
NSG1P:NSG1:GFP fusion protein was observed directly in the complementary 368
transgenic lines, which confirmed by immunoblotting with anti-GFP (Supplemental 369
Figure 7C). The GFP signals were localized in the nucleus, which suggested that NSG1 is 370
a nucleus-targeted protein (Figure 5B). Next, a dual luciferase reporter was used to 371
investigate further the transcriptional regulation activity of NSG1 (Figure 5C-5E). The 372
positive control VP16 showed relatively high luciferase activity, whereas VP16-NSG1 373
fusion protein displayed much lower activity than the positive control (Figure 5D). 374
Meanwhile, the full-length NSG1 protein and the NSG1-C (129–175) truncated protein 375
(containing Motif 2) showed significantly lower luciferase activity than the negative 376
control, whereas the luciferase activity in NSG1-N (1-40) (containing Motif 3), NSG1-377
△N (41-175) (lacking Motif 3), and NSG1-△C (1-135) (lacking of Motif 2), showed no 378
significant difference compared with the negative control (Figure 5E). These findings 379
demonstrated that NSG1 acted as a transcriptional repressor and that the C-terminal EAR 380
motif, but not the N-terminal motif, was necessary for activity of transcriptional 381
repression. 382
383
13
Expression Patterns of NSG1 384
To determine the spatiotemporal expression patterns of NSG1, we used the GFP 385
reporter gene to detect NSG1 expression in panicles at different developmental stages. In 386
the complementation transgenic lines, the GFP expression pattern in the young panicle 387
was examined in detail. Strong GFP signals were observed in the primordia of the 388
rudimentary glume and sterile lemma (Figures 6A–6D), while very weak GFP signal was 389
detected in lemma and palea (Figures 6C and 6D). With spikelet development, the signal 390
in both the sterile lemmas and rudimentary glumes was still high, although it was stronger 391
in the latter (Figures 6E–6H). At a stage after about Sp7, asymmetrical signal distribution 392
was observed in sterile lemmas, in which GFP expression was stronger in the basal 393
margin region but weaker expression was observed in other regions (Figure 6G). In 394
addition, after about Sp7 stage, the GFP signal was obvious at the apex of the lemma and 395
palea primordia and in the mrp primordia (Figures 6G). 396
These results indicate that during spikelet development, NSG1 was always strongly 397
expressed in the rudimentary glumes and sterile lemma, while the level of its expression 398
in mrp and other organs/regions rose gradually. Therefore, the NSG1 expression pattern 399
corresponded well with the function of the protein in organ identity of the spikelet. 400
401
Effects of NSG1 Mutation on Expression Patterns of Organ Identity Genes during 402
Early Stages of Spikelet Development 403
To further examine the regulatory mechanism of NSG1, the expression patterns of four 404
genes responsible for the specification of organ identity in spikelet were investigated 405
during the early stages of spikelet development (from stages Sp4 to Sp8; Figure 7; 406
Supplemental Figure 9). Quantitative real-time PCR (qPCR) analysis showed that 407
transcription of DL was up-regulated in the nsg1-1 mutant during the three defined stages 408
of spikelet development (panicle length <0.5 cm, 0.5–1 cm, and 1–2 cm), LHS1 was up-409
regulated in the nsg1-1 mutant in >0.5cm panicles, and MFO1 transcripts were more 410
abundant in 0.5–1 cm panicles of the nsg1-1 mutant, compared with those of the WT 411
(Figures S8A-S8C). Transcripts of G1 exhibited a significant decrease in abundance in 412
the nsg1-1 mutant at all defined stages of spikelet development compared with those in 413
the WT (Figures S8D). 414
14
Expression of these organ identity genes was further detected using in situ 415
hybridization. We firstly detected the expression pattern of LHS1, the ectopic expression 416
of which resulted in lemma-like sterile lemma and elongated rudimentary glumes in rice 417
in previous study (Wang et al., 2017). In WT spikelets, the strong LHS1 signal was 418
always detected in lemma and palea primordia after about Sp4 (Figures 7A-7D). It is 419
noteworthy that LHS1 was mainly expressed in the bp of palea, but not in the mrp (Figure 420
7I and 7M). In nsg1-1 spikelet, the expression of LHS1 in lemma primordia was not 421
changed. However, the ectopic expression of LHS1 could be detected in the sterile lemma 422
primordia frequently and in the mrp primordia occasionally (Figure 7E-7H, 7J-7L and 423
7N-P). To further clarify the ectopic expression domain, we conducted the in situ 424
hybridization of LHS1 using transverse sections of WT and nsg1-1 spikelets at Sp8 stage. 425
It was very clear that LHS1 was expressed strongly in the inner lemma-like sterile lemma 426
of nsg1-1 spikelets, and obvious signals were also detected in mrp of nsg1-1 palea 427
(Figure 7J-7L and 7N-P), yet there were no signal detected in these regions of WT 428
spikelets (Figure 7I and 7M). We also found that LHS1 was expressed in whole domain 429
of some lemma-like palea of nsg1-1 spikelets, in which it was difficult to distinguish the 430
mrp and bp of palea (Figure 7L and 7P). Taken together, these results indicated that LHS1 431
was ectopically expressed in sterile lemma and mrp of nsg1-1 spikelets frequently, and in 432
rudimentary glumes occasionally. 433
The expression patterns of G1, DL and MFO1 were also detected by in situ 434
hybridization. Strong signals for G1 were detected in WT sterile lemmas during 435
primordium initiation and formation at approximately Sp4, then slowly decreased but 436
were still detectable in sterile lemmas after initiation of elongation during stages Sp5 to 437
Sp8 (Figures S9A). A signal for G1 expression was also observed in the primordia of the 438
rudimentary glumes and palea (Figures S9A2–S9A4). In spikelets of the nsg1-1 mutant, 439
however, the G1 expression signal was observed only in a minority of palea primordia 440
and was almost undetectable in the primordia of the sterile lemmas and rudimentary 441
glumes at all stages examined (Supplemental Figure 9B). During stages Sp4 and Sp5, DL 442
expression in the WT was detected only in lemma primordia and not in pistil primordia 443
(Figures S9C1 and S9C2). After Sp6, DL was also expressed in pistil primordia in the 444
WT (Figures S9C3–S9C5). In nsg1-1 spikelets, DL expression in primordia of the lemma 445
15
and pistil was unchanged, and ectopic expression signals of DL were detected in 446
primordia of the sterile lemmas and palea (Supplemental Figure 9D, indicated by red 447
type). During Sp4 to Sp8, MFO1 expression was detected in the primordia of the mrp and 448
lodicule in both the WT and nsg1-1 mutant (Figures S9E and S9F). However, ectopic 449
expression of MFO1 was detected in the partial primordia of the sterile lemmas and 450
rudimentary glumes (Supplemental Figure 9F, indicating by red type). In addition, the 451
region of MFO1 expression in the FM was changed in nsg1-1 spikelets. During Sp4, 452
MFO1 expression was restricted to the FM of the WT (Supplemental Figure 9E1), 453
whereas MFO1 expression was expanded to the receptacle and rachilla in spikelets of the 454
nsg1-1 mutant (Supplemental Figure 9F1-F5, indicated by red triangles). 455
In summary, ectopic expression of LHS1, DL, and MFO1, and absence of G1 456
expression in primordia of the rudimentary glumes, sterile lemmas, and palea of nsg1-1 457
spikelets implied that the expression of these genes was regulated negatively or positively 458
by NSG1. 459
460
NSG1 represses LHS1 directly by interacting with co-repressors OsTPRs 461
The above analysis of EAR motifs, transcriptional activity and expression patterns 462
indicates that NSG1 may act as a repressor to regulate the transcription of DL, LHS1, 463
and/or MFO1. It was found firstly that there was a cluster of DBS-like motifs located in 464
the LHS1 promoter (Figures 8A), which were characterized as a conserved binding site of 465
C2H2 zinc finger domain (Huang et al., 2009). Then, chromatin immunoprecipitation 466
(ChIP) assays were used to examine whether NSG1 protein is able to bind to this region 467
of the LHS1 promoter. Chromatin isolated from young panicles of NSG1P:NSG1:GFP 468
transgenic lines were immunoprecipitated with the GFP antibody and then subjected to 469
qPCR analysis using primer P1. The repeatable results showed that NSG1P:NSG1:GFP 470
protein was able to bind stably to this P1 site (Figures 8B). These results suggest that 471
NSG1 protein might be responsible for direct regulation of LHS1. Next, we detected the 472
effect of NSG1 protein on the expression of the firefly luciferase (LUC) gene reporter 473
using the DBS-like motif region of LHS1 as the promoter for transient expression assays 474
in Nicotiana benthamiana leaves. Compared with the negative control p35Sm:LUC 475
reporter, the LUC activity was significantly increased with the pLHS1P-35Sm:LUC 476
16
reporter (Figure 8C). Co-expression of the pLHS1P-35Sm:LUC reporter with 477
35S:NSG1WT led to a significant decrease of the LUC activity, whereas 35S:NSG1nsg1-1 478
failed to down-regulate the expression of the pLHS1P-35Sm:LUC reporter (Figures 8C). 479
Therefore, these results indicated that NSG1WT protein had the ability to repress the 480
expression of LHS1 by binding the DBS-like motif region in LHS1 promoter. 481
The proteins with EAR motifs are generally believed to interact with co-repressors 482
TPL/TPR. In rice, there are three TPR proteins, OsTPR1 (LOC_Os01g15020), OsTPR2 483
(LOC_Os08g06480, also named as ABERRANT SPIKELET AND PANICLE1, ASP1), and 484
OsTPR3 (LOC_Os03g14980) (Yoshida A et al., 2012). To further clarify the molecular 485
mechanism of NSG1 regulating LHS1 expression, the interaction between NSG1 and 486
OsTPRs was examined. Firstly, Y2H assays were performed to determine the physical 487
interaction between NSG1 and OsTPRs. NSG1 interacted with all three OsTPR proteins 488
in yeast cells (Figure 8D). Next, a BiFC assay was conducted to detect whether the NSG1 489
could interact with OsTPRs in N. benthamiana leaves. The YFP signal was found in the 490
nucleus of epidermal cells of N. benthamiana leaves co-expressing the NSG1-YC fusion 491
protein with YN-OsTPR1, YN-OsTPR2, or YN-OsTPR3 (Figure 8E). Thus, NSG1 can 492
interact with OsTPRs in plant cells. It is well known that TPL/TPR can interact with 493
HDACs to mediate the acetylation level of histone protein in the targeted region (Ke et 494
al., 2015). Therefore, we further detected the histone acetylation level in the LHS1 495
promoter and CDS region by ChIP-qPCR using the chromatin immunoprecipitated by an 496
anti-H3K9ac antibody. The acetylation levels of H3K9 at P2, P4, P5, P7, P9 and P12 497
sites, most of them in the first intron of LHS1, were significantly increased in the nsg1-1 498
mutant compared with those in the WT (Figure 8F). 499
Therefore, the above-described results support that NSG1 can directly bind to the 500
promoter or other regulating region of LHS1 genes, then recruit the co-repressor TPRs to 501
repress LHS1 expression by down-regulating the acetylation levels of histone on 502
chromosomes at LHS1 (Figure 8G). 503
504
Genetic interaction between NSG1 and LHS1 505
We further examined genetic interactions between NSG1 and LHS1 using nsg1-1 lhs1-z 506
double mutants. The lhs1-z mutant is an allelic loss-of-function mutant of LHS1, as 507
17
characterized by our group (Li, 2008). The mutation of LHS1 in lhs1-z and other allelic 508
mutants led to transformation of the lemma into “leaf-like organs”, which were longer 509
and thinner than the WT lemma, owing to the absence or decreased abundance of fibrous 510
sclerenchyma and the presence of a weaker-silicified abaxial epidermis. However, the 511
identities of the sterile lemmas and rudimentary glumes were not affected (Figure 9A; 512
Jeon et al., 2000; Prasad et al., 2005). In the double mutant, the sterile lemmas and 513
rudimentary glumes remained longer and wider than those of the WT or lhs1-z, but were 514
distinctly shorter and thinner than those of the nsg1-1 mutant (Figures 1A1–1A3, 9A1–515
9A3, and 9B1–9B3). It was also observed that the abaxial surface of the inner 516
rudimentary glumes and the outer and inner sterile lemmas were smooth, with several 517
trichomes in the double mutant (Figures 9B4–9B6), which were identical to the sterile 518
lemmas of the WT and lhs1-z mutant (Figures 1A4, 1A5, 9A4, and 9A5). 519
Histological analysis revealed that very few fibrous sclerenchyma cells and silicified 520
abaxial epidermis cells were observed in the rudimentary glumes and sterile lemmas of 521
the double mutant (Figures 9B7–9B9), whereas numerous such cells were observed in 522
some rudimentary glumes and sterile lemmas of the nsg1-1 mutant (Figures 2B–2D). In 523
addition, no rudimentary glumes were transformed into lemma-like organs in the double 524
mutant, and the number of lemma-like sterile lemmas was distinctly less than that in the 525
nsg1-1 mutant (Figure 9C). 526
The expression levels of LHS1, DL, and MFO1 were detected by qPCR in rudimentary 527
glumes and sterile lemmas of the double mutant as well as the lhs1-z and nsg1-1 single 528
mutants (Figures 9D–9F). Give that lhs1-z was a transposon insertion mutant of LHS1, 529
expression of LHS1 was not detected in the lhs1-z single mutant nor the nsg1-1 lhs1-z 530
double mutant (Figure 9D). In contrast to the ectopic expression of DL in the outer and 531
inner sterile lemmas of the nsg1-1 spikelet, DL expression was not detected in the outer 532
sterile lemma and was substantially decreased in the inner sterile lemma of the double 533
mutant, and was not detected in these organs in the lhs1-z mutant (Figure 9E). MFO1 was 534
weakly expressed in rudimentary glumes and sterile lemmas of the lhs1-z mutant, similar 535
to that of the WT, whereas the ectopic expression level was higher in the inner 536
rudimentary glume, and in the outer and inner sterile lemmas of the double mutant than in 537
the nsg1-1 single mutant (Figure 9F). Collectively, these findings suggest that ectopic 538
18
LHS1 activation was an important cause of ectopic formation of lemma-like tissue in the 539
nsg1-1 sterile lemmas and rudimentary glumes. 540
541
DISCUSSION 542
The Role of NSG1 in Specification of Sterile Lemmas and Rudimentary Glumes 543
In three allelic mutants of NSG1, the rudimentary glumes and the sterile lemmas were 544
elongated and/or widened, and most of rudimentary glumes and a minority of outer sterile 545
lemmas were transformed into the lemma and/or mrp-like organ, and most of outer sterile 546
lemmas and inner sterile lemmas were transformed into the lemma -like organ. It was 547
previously reported that G1 regulates sterile lemma identity. In all mutants of G1, the 548
sterile lemma is transformed into a lemma-like organ (Yoshida et al., 2009). PAP2 549
regulates the specification of sterile lemmas and rudimentary glumes. In mutants of 550
PAP2, the sterile lemmas and rudimentary glumes are elongated and show a leaf/lemma-551
like identity (Gao et al., 2010). These results suggest that G1, PAP2, and NSG1 are 552
involved in maintaining the identity of rudimentary glumes and/or sterile lemmas by 553
preventing incorrect cell differentiation. 554
Two prevailing hypotheses on the origin and evolution of the sterile lemmas have been 555
proposed. One hypothesis states that a putative ancestor of Oryza had a “three-florets 556
spikelet” that contained a terminal floret and two lateral florets, in which the palea and 557
inner floral organs were absent, and the residual lemma was reduced to a sterile lemma 558
during evolution (Arber, 1935; Kellogg, 2009; Yoshida et al., 2009; Kobayashi et al., 559
2010; Zhang et al., 2017). The second hypothesis suggests that the spikelet of Oryza 560
species contained only one floret, and the sterile lemmas and rudimentary glumes are 561
universally regarded as severely reduced bract structures (Schmidt and Ambrose, 1998; 562
Hong et al., 2010). Mutation of G1, EG1, PAP2, ASP1, or NSG1, or ectopic expression of 563
LHS1 causes homeotic transformation of the sterile lemma into a lemma to various 564
degrees, which suggests that the sterile lemmas are homologous to lemmas, and thus 565
partially supports the first hypothesis (Li et al., 2009a; Yoshida et al., 2009; Yoshida et al., 566
2012; Wang et al., 2013; Lin et al., 2014; Wang et al., 2017). In the lf1 mutant, lateral 567
florets are formed in the axil of each sterile lemma, which provides strong support for the 568
“three-florets spikelet” hypothesis (Zhang et al., 2017). However, the sterile lemma is 569
19
degenerated and acquires the identity of a rudimentary glume in the mfs1 mutant. 570
Interestingly, some elongated rudimentary glumes appeared to acquire the identity of a 571
sterile lemma and/or lemma in the nsg1 mutants. These findings also support the opinion 572
of sterile lemmas being homologous to rudimentary glumes in the second hypothesis. 573
In most grass species, the spikelet lacks sterile lemma-like organs, and contains one 574
or more florets and bract-like glume organs, which are considered to be equivalent to the 575
rudimentary glumes of Oryza species (Yoshida et al., 2009; Hong et al., 2010). The bract-576
like glume organ resembles the lemma in size and structure in some grass species, such as 577
maize and wheat (Kellogg, 2001; Yoshida et al., 2009), whereas it is severely reduced in 578
Oryza species (Bommert et al., 2005; Li et al., 2009b). The above results therefore imply 579
that in rice the sterile lemma was derived from the lemma of the lateral floret, and the 580
sterile lemmas and lemma are homologous to the rudimentary glume, which are derived 581
from bract-like structures. 582
Interestingly enough, according to the phenotype analysis of nsg1-1 and nsg1-3 583
mutants, the outer rudimentary glume was absent in approximately 100% of nsg1-1 584
spikelets and 78% of nsg1-3 spikelets (Figure 1G; Supplemental Figure 3A). In fact, in 585
previously published research, we found the outer rudimentary glume was invisible at 586
later spikelet growth stage under optical microscopy (Wang et al., 2013), but could not 587
clarify whether it was degraded or absent. In this study, to further conform this, SEM 588
analysis was used to carefully investigate the formation and differentiation of organ 589
primordia in nsg1-1 spikelets at an early development stage. The results clearly indicated 590
that the primordia of outer rudimentary glumes were not formed from the beginning 591
(Figures 3B and 3C). There was degeneration on the glume-like organs in many rice 592
mutants, such as lemma and palea in lhs1 and sl1 mutants, sterile lemma in mfs1 mutant 593
(Jeon et al., 2000; Prasad et al., 2005; Ren et al., 2013; Xiao et al., 2009). However, the 594
absence of all parts of an organ in spikelets is by no means rare. Therefore, this mutant 595
will be useful in the future to clarify the formation mechanism of outer rudimentary 596
glumes. 597
598
NSG1 Regulates Palea and Lodicule Development 599
In grass florets, the palea was considered to show an identity and origin distinct from 600
20
those of the lemma. In general, the palea is considered to be homologous to the prophyll 601
(the first leaf produced by the axillary meristem) formed on a floret axis, whereas the 602
lemma corresponds to the bract (the leaf subtending the axillary meristem) formed on a 603
spikelet axis (Kellogg, 2001; Ohmori et al., 2009). 604
Several previous studies have indicated that the specification of mrp and bp in the 605
palea is controlled by different genes. In the depressed palea1 (dp1) mutant, the bp is lost 606
and two mrp-like structures remain (Luo et al., 2005; Jin et al., 2011). In the retarded 607
palea1 (rep1) mutant, development of the bp is delayed (Yuan et al., 2009). In the mfs1-1 608
mutant, the bp is degenerated in most florets and is absent in a minority of florets. 609
However, in mutants of CHIMERICAL FLORAL ORGANS 1 (CFO1) and MFO1, the mrp 610
is transformed into a lemma-like structure (Ohmori et al., 2009; Li et al., 2010; Sang et 611
al., 2012). Therefore, whilst MFS1, DP1, and REP1 determine bp identity, MFO1 and 612
CFO1 are involved in regulation of mrp identity. In the nsg1-1 mutant, over 50% of 613
spikelets contained a lemma-like palea because the mrp gained a lemma-like identity to 614
various degrees, and approximately one-third of spikelets showed a degenerated palea 615
owing to degeneration or absence of the bp. These results indicate that NSG1 plays a dual 616
role in regulation of both bp and mrp development in the palea, possibly through 617
regulation of genes in different pathways. 618
The lodicule in grasses is usually considered to be the equivalent organ to the petal of 619
dicots, which is mainly controlled by B-function genes. The rice B-class mutant 620
superwoman1 (spw1/lhs16) and OsMADS2 OsMADS4 double RNAi plants show 621
transformation of the lodicules into organs that resemble the mrp (Nagasawa et al., 2003; 622
Yadav et al., 2007; Yao et al., 2008). The mutants of CFO1 and MFO1 show 623
lemma/pistil-like but not mrp-like lodicules. It has been demonstrated that CFO1 624
regulates lodicule identity by restricting the expression of DL, which is involved in 625
specification of the lemma and pistil. Therefore, specification of the lodicule in rice 626
involves different regulatory pathways associated with B-function genes and CFO1–627
MFO1 genes (Li et al., 2010; Sang et al., 2012). In the present study, the lodicules were 628
elongated and/or widened, and gained a similar identity to that of the mrp or the lemma in 629
the nsg1 mutants. These results suggest that NSG1 is involved in the regulation of 630
lodicule identity associated with more B-function gene pathways than the CFO1–MFO1 631
21
pathway. 632
633
NSG1 Maintains Organ Identity of the Spikelet by Regulation of LHS1, MFO1, DL, 634
and G1 Expression 635
In rice spikelets, the organs can be divided into floral organs (lemma, palea, lodicule, 636
stamens, and pistil) within the floret and non-floral organs (rudimentary glumes and 637
sterile lemmas) that subtend the floret. The DL gene and some MADS-box genes, 638
including ABCE-class genes (A-class RICE APETALA 1A, B-class OsMADS2/4/16, C-639
class RICE AGAMOUS and OsMADS58, D-class OsMADS13, and E-class LHS1), MFO1, 640
and CFO1, are considered to be floral organ identity genes (Li et al., 2011; Sang et al., 641
2012). The genes G1, EG1, PAP2, and ASP1 are involved in specification of the 642
rudimentary glume and sterile lemma (Yoshida et al., 2009; Gao et al., 2010; Yoshida et 643
al., 2012). The limited proper expression domain of these genes is necessary to maintain 644
these organ identities. The interactive restriction between SPW1 and DL might be crucial 645
for specification of stamens and the pistil (Nagasawa et al., 2004; Yamaguchi et al., 646
2004), and repression of CFO1 to DL is crucial for formation of the mrp, lodicule, and 647
stamens (Sang et al., 2012). Epigenetic regulation by the EMF1-like gene, DEFORMED 648
FLORAL ORGAN1 (DFO1)/CURVED CHIMERIC PALEA 1 (CCP1), which is widely 649
responsible for repression of OsMADS3, OsMADS58, OsMADS13, and DL, also 650
contributes to establishment of the identity of floral organs, such as the palea, lodicule, 651
and stamen (Yang, et al., 2015; Zheng, et al., 2015). However, limited information is 652
available on gene regulation pathways for non-floral organs of the spikelet in rice. 653
In the present study, the identities of all organs except the pistil and lemma were affected 654
in the nsg1-1 spikelet and were all transformed into lemma/mrp-like organs 655
to various degrees. In the nsg1-1 spikelet, LHS1, DL, and MFO1 were ectopically 656
expressed in two or more organs, including the rudimentary glume, sterile lemma, palea, 657
lodicule, and stamen, whereas G1 was expressed in the rudimentary glume and sterile 658
lemma. We further verified that the NSG1 protein could bind to the sites in the promoter 659
of LHS1, interact with rice co-repressors OsTRPs, and then recruit HDACs to mediate the 660
histone acetylation level in the targeted region. Collectively, these results suggest that 661
NSG1 maintained organ identities in the spikelet by direct epigenetic repression of LHS1, 662
22
and indirect regulation of DL, MFO1 and G1 (Figure 10). Following loss of function of 663
NSG1 in the nsg1 mutants, these organ identity genes were ectopically expressed or down-664
regulated, which led to the transformation of the organs into lemma-like organs (Figure 665
10). 666
667
METHODS 668
Plant Materials 669
Two mutants of rice (Oryza sativa), nsg1-1 and nsg1-3, were identified from among 670
ethylmethane sulfonate-treated plants of the wild-type xian-type (indica) restorer line 671
‘Jinhui 10’ (J10) and the wild-type xian-type (indica) maintainer line ‘Xida1B’ (1B), 672
respectively. An additional mutant, nsg1-2, was derived from a T-DNA insertion library 673
(Rice Mutant Database RMD) (Zhang et al., 2007) and had the geng-type (japonica) 674
background of ‘Zhonghua11’ (ZH11). All plants were cultivated in paddies at 675
Chongqing, China. 676
677
Microscopy 678
Panicles were collected at different developmental stages and fixed in FAA (50% 679
ethanol, 0.9 M glacial acetic acid, and 3.7% formaldehyde) over 16 h at 4ºC. The fixed 680
samples were dehydrated with a graded ethanol series, infiltrated with xylene, and 681
embedded in paraffin (Sigma). The 8-μm sections were transferred onto poly-L-lysine-682
coated glass slides, deparaffinized in xylene, and dehydrated through an ethanol series. 683
The sections were stained sequentially with 1% safranin O (Amresco) and 1% Fast Green 684
(Amresco), then dehydrated through an ethanol series, infiltrated with xylene and finally 685
mounted beneath a cover slip. Light microscopy was performed using an Olympus BX53 686
microscope. For scanning electron microscopy, fresh samples were examined using a 687
Hitachi SU3500 scanning electron microscope with a −20ºC cool stage. The stages of 688
early spikelet development were identical to those defined previously (Ikeda et al., 2004). 689
690
RNA Isolation and Reverse Transcription qPCR Analysis 691
Total RNA was isolated from young panicles and organs within the spikelet from 692
mutants and the WT using the RNAprep Pure Plant RNA Purification Kit (Tiangen). The 693
23
first-strand cDNA was synthesized from 2 µg total RNA using oligo(dT)18 primers in a 25 694
µL reaction volume using the SuperScript™ III Reverse Transcriptase Kit (Invitrogen). 695
Half a microliter of the reverse-transcribed RNA was used as the PCR template with 696
gene-specific primers (Supplemental Table 1). Quantitative real-time PCR analysis was 697
performed with a CFX96 Real-Time PCR Detection System (Bio-Rad) and the TB 698
Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (RR820A) (TaKaRa). PCR cycling 699
conditions for amplification were 95℃ for 30s followed by 40 cycles of 95℃ for 5s, 700
60℃ for 30s. Relative expression levels were determined compared with wild-type levels 701
using the 2(-ΔCt) analysis method. ACTIN was used as an endogenous control. At least 702
three replicates were performed, from which the mean value was used to represent the 703
expression level. 704
705
Complementation Test 706
For the complementation test, the WT genomic fragment of NSG1 (Os04g36650), 707
which consisted of the 2,925 bp upstream sequence from the start codon and 525 bp 708
coding sequence, was amplified using the primers NSG1com-F2 and NSG1com-R1. 709
Given that the GC content in the fragment was extremely high, the high-fidelity 710
thermostable DNA polymerases KOD FX and KOD neo (TOYOBO, Shanghai, China) 711
were used to amplify the fragment of NSG1 (Os04g36650). The intermediate vector 712
35S:GFP (S65T):NOS (pCAMBIA1301) was digested using HindIII and BamHI to 713
generate the construct pCAMBIA1301:GFP:NOS. Using the method of homologous 714
recombination, the resulting PCR products were inserted into the vector framework 715
pCAMBIA1301:GFP:NOS to generate the complementary vector pCAMBIA1301-716
NSG:GFP:NOS (NSG:GFPcom). The recombinant plasmids were introduced into the 717
nsg1-1 mutant using the Agrobacterium-mediated transformation method as described 718
previously (Sang et al., 2012). 719
For examination of GFP fluorescence in the transgenic plants, young panicles and 720
other tissues were transferred to a glass slide, then examined under an Olympus MVX10 721
microscope and an Olympus FluoView 1000-Confocal laser scanning microscope. The 722
primer sequences are listed in Supplemental Table 1. 723
724
24
DNA Gel Blot Analysis 725
For DNA gel blot detection, genomic DNA (15–20 μg) extracted from J10, XD1B, 726
ZH11, and Nipponbare was used. The plasmid NSG:GFPcom was linearized with EcoRI 727
as a positive control. The probes of NSG1 promoter and its open reading frame (ORF) 728
were amplified using the primer pairs NSG1sb-PF and NSG1sb-PR, and NSG1sb-OF and 729
NSG1sb-OR, respectively, and labeled using the PCR DIG Probe Synthesis Kit (Roche), 730
in accordance with the manufacturer’s recommendations. Transfer to the membrane was 731
performed using the iBlotTM 2 Gel Transfer Device (Invitrogen). Prehybridization, 732
hybridization, and washing were performed in accordance with the manufacturer’s 733
recommendations and as reported by Southern (Alwine et al., 1977). Detection was 734
performed using the DIG-High Prime DNA Labeling and Detection Starter Kit II 735
(chemiluminescent detection), in accordance with the manufacturer’s recommendations. 736
737
Protein Sequence and Phylogenetic Analysis 738
Protein sequences were obtained by searching the GenBank database 739
(http://www.ncbi.nlm.nih.gov/genbank/) using the NSG1 protein sequence as a query. 740
Using MEGA 5.0, a phylogenetic tree was constructed using the neighbor-joining method 741
based on the Jones–Taylor–Thornton matrix-based model. Bootstrap support values for 742
the tree topology were calculated from 1000 replicates (Saitou and Nei, 1987; 743
Felsenstein,1985; Jones et al., 1992; Tamura et al., 2011). Protein motifs were predicted 744
using MEME (http://alternate.meme-suite.org/). 745
746
Analysis of Transcriptional Activity 747
Using the dual luciferase reporter (DLR) assay system, we analyzed the transcriptional 748
activity of NSG1 in rice protoplasts. The DLR assay system was used with a GloMax 20-749
20 luminometer (Promega) to measure the relative luciferase activity (Ren et al., 2018). 750
The coding frame of the NSG1 cDNA was fused to the GAL4 DNA-binding domain 751
(BD), driven by the 35S promoter. VP16, a transcriptional activator, was used as a 752
positive control and GAL4-BD was regarded as a negative control. The VP16, VP16-753
NSG, BD-NSG1, and GAL4-BD effectors were transiently expressed in rice protoplasts. 754
The primers used are listed in Supplemental Table 1. 755
25
756
In Situ Hybridization 757
The gene-specific NSG1 probe was amplified with the primers NSG1-HF and NSG1-758
ishSP6HR and labeled using the DIG RNA Labeling Kit (SP6/T7) (Roche). Probes for 759
known floral organ genes were prepared using the same method. Pretreatment of sections, 760
hybridization, and immunological detection were performed as described previously 761
(Sang et al., 2012). The primer sequences are listed in Supplemental Table 1. 762
763
Protein Interaction Analyses 764
Yeast two-hybrid (Y2H) assays were performed using the Match-maker TM Gold 765
Yeast Two-Hybrid system (Clontech, MountainView, CA, USA). The full-length coding 766
region of NSG1 was amplified and ligated into the yeast expression vector pGADT7 767
(Clontech) to produce pGADT7-NSG1. The full-length coding region of TPRs were 768
introduced into pGBKT7 (BD) (Clontech) to produce pGBKT7-TPRs. The yeast strain 769
used in Y2H assays was Y2HGOLD. The pGADT7-T+pGBKT7-lam as negative control 770
and the pGADT7-T+pGBKT7-53 as positive control. These plasmids were co-771
transformed into Y2HGOLD strain in an AD-BD-coupled manner. Detailed procedures 772
were described in the manufacturer’s instruction (Yeast Protocols Handbook; PT3024-1, 773
Clontech). To conduct BiFC assays, NSG and TPRs were amplified by specific primers 774
(Supplemental Table 1) with KOD-neo polymerase and then ligated into BiFC vectors, 775
including pSCYNE (SCN, modified) and pSCYCE (SCC, modified) (Waadt et al., 2008). 776
Then, vector pairs were co-transformed into N. benthamiana leaves. The fluorescence 777
signals were captured under a confocal laser scanning microscope (Zeiss LSM 800). 778
779
ChIP-qPCR 780
NSG1:GFPcom, WT, and mutant plants were used in the ChIP examination. Young 781
panicles (<2 cm) were collected for isolation of nuclear extracts. The EpiQuik™ Plant 782
ChIP Kit (Epigentek, P-2014-48), anti-GFP antibody (Abcam, ab290, ChIP Grade), and 783
anti-Histone H3 (acetyl K9) antibody (Abcam, ab10812, ChIP Grade) were used for ChIP 784
assays. All PCR experiments were conducted using 40 cycles of 95℃ for 5s, 60℃ for 785
30s, and 72℃ for 30s in a reaction mixture containing 10 pmol of each primer and 1μl 786
26
DNA from ChIP or control or 1μl of input DNA diluted 20-fold (per biological replicate) 787
as template. More than three biological repeats (1 g panicle sample each) with three 788
technical repeats each were used to produce data for statistical analysis. Experimental 789
procedures for ChIP-qPCR were performed as described previously (Xu et al., 2010). The 790
primer sequences are listed in Supplemental Table 1. 791
792
Transient Expression Regulation Assays in N. benthamiana Leaves. 793
The LHS1 promoter and a mini CaMV35S promoter were amplified with the primer 794
pairs in Supplemental Table 1 and cloned into pGreenⅡ0800-LUC double-reporter 795
vector. The full-length coding region of NSG1 from J10 and nsg1-1 mutant were PCR 796
amplified with gene-specific primers (Supplemental Table 1) and then recombined with 797
the vector pCAMBIA1300 (Cambia) with two CaMV35S promoters to generate the 798
2x35Spro:NSG1WT and 2x35Spro:NSG1nsg1-1constructs. The above constructs were then 799
transformed into Agrobacterium tumefaciens strain GV3101. The Agrobacterium strains 800
containing different constructs were incubated, harvested, and resuspended in infiltration 801
buffer (10 mM MES, 0.2 mM acetosyringone, and 10 mM MgCl2) to an ultimate 802
concentration of OD600 = 0.6. Equal volumes of different combinations of Agrobacterium 803
strains were mixed and coinfiltrated into N. benthamiana leaves using a needleless 804
syringe. Plants were placed at 25°C for 48 h. 805
LUC and REN luciferase activities were measured using the Dual Luciferase Assay Kit 806
(Promega). The analysis was executed using the Luminoskan Ascent microplate 807
luminometer (Thermo) according to the instructions of the manufacturer. The results 808
were calculated by the ratio of LUC/REN (Ba et al., 2016). At least six transient assay 809
measurements were made for each assay. 810
811
Accession Numbers 812
Sequence data from this article can be accessed in the GenBank database under the 813
following accession numbers: The GenBank accession numbers for the cDNA sequences 814
of NSG, G1, DL, LHS1, MFO1, OsTPR1, OsTPR2 and OsTPR3 are GQ999998, 815
AB512480, AB106553, NM_001055911, FJ666318, AP014957, AK111830 and 816
AP014959 respectively. Locus identifications in the Rice Genome Annotation Project 817
27
Database are as follows: NSG1 (LOC_Os04g36650), G1 (LOC_Os07g04670), DL 818
(LOC_Os03g11600), LHS1 (LOC_Os03g11614), MFO1 (LOC_Os02g45770), OsTPR1 819
(LOC_Os01g15020), OsTPR2 (LOC_Os08g06480), and OsTPR3 (LOC_Os03g14980); 820
BAC OSJNBa0058G03 (OSJNBa0058G03), and BAC OSJNBb0056O04 821
(OSJNBb0056O04). 822
28
Supplemental Data 823
Supplemental Figure 1. Structures of Cell Layers of Partial Organs in Spikelets of the 824
Wild Type and nsg1-1 Mutant. 825
Supplemental Figure 2. Phenotypes of Spikelets in the Wild Type and the nsg1-2 and 826
nsg1-3 Mutants. 827
Supplemental Figure 3. Percentage of Elongated or Lemma-like Organs and Relative 828
Expression Levels of Organ Identity Genes in Spikelets of the nsg1-2 and nsg1-3 829
Mutants. 830
Supplemental Figure 4. NSG1 Expression in the nsg 1-2 Mutant. 831
Supplemental Figure 5. DNA Gel Blot of NSG1 in the Genome of Four Rice Cultivars. 832
Supplemental Figure 6. Protein Sequence Analysis of Candidate Gene in Wild type and 833
nsg1 Mutants. 834
Supplemental Figure 7. Protein Motif Analysis of NSG1-like Protein and Immunoblots 835
in GFP-fusion Transgenic Plants. 836
Supplemental Figure 8. qPCR analysis of Organ Identity Genes in Spikelets of the Wild-837
type and the nsg1-1 Mutant. 838
Supplemental Figure 9. Expression Pattern of Organ Identity Genes in Spikelets of the 839
Wild-type and the nsg1-1 Mutant. 840
Supplemental Figure 10. Promoter Sequence of LHS1 Highlighting the DBS-like Motif. 841
Supplemental Table 1. Primers used in this study. 842
Supplemental Data set 1. NSG1 tree.fas alignment file 843
Supplemental Data set 2. NSG1 tree.meg alignment file 844
Supplemental Data set 3. NSG1 tree.mts alignment file 845
ACKNOWLEDGMENTS 846
This work was supported by the National Natural Science Foundation of China 847
29
(31730063, 31271304, and 31971919), National Key Program for Research and 848
Development (2017YFD0100202), Project of CQ CSTC (CSTC2017jcyjBX0062), and 849
Graduate Student Scientific Research Innovation Projects in Chongqing (CYS2015066), 850
and the Fundamental Research Funds for the Central Universities (XDJK2016A013). We 851
are very grateful to Prof. Keming Luo (College of Life Sciences, Southwest University) 852
for kindly providing the pGreenⅡ0800-LUC double-reporter vector, pSCYNE (SCN, 853
modified) and pSCYCE (SCC, modified) vectors. 854
855
AUTHOR CONTRIBUTIONS 856
Y.-F. L. and G.-H. H. designed the research. H. Z. performed ChIP analysis. H. C. and H. 857
Z. performed the mapping-based clone and phenotype analysis. H.-L. W., T. Z., H.Z. and858
X.-Q. Z. performed the analysis of the expression pattern and protein activation. Z.-W.W. 859
performed the vector construction. The other authors contributed data analysis. Y.-F. L, H. 860
Z. and T. Z. wrote the manuscript.861
862
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Yoshida, A., Ohmori, Y., Kitano, H., Taguchi-Shiobara, F., and Hirano, H.Y. (2012). 1041
ABERRANT SPIKELET AND PANICLE1, encoding a TOPLESS-related 1042
transcriptional co-repressor, is involved in the regulation of meristem fate in rice. 1043
Plant Journal 70, 327-339. 1044
Yoshida, A., Sasao, M., Yasuno, N., Takagi, K., Daimon, Y., Chen, R.H., Yamazaki, R., 1045
34
Tokunaga, H., Kitaguchi, Y., Sato, Y., Nagamura, Y., Ushijima, T., Kumamaru, 1046
T., Iida, S., Maekawa, M., Kyozuka, J. (2013). TAWAWA1, a regulator of rice 1047
inflorescence architecture, functions through the suppression of meristem phase 1048
transition. P Natl Acad Sci USA 110 (2), 767-772. 1049
Yuan, Z., Gao, S., Xue, D.W., Luo, D., Li, L.T., Ding, S.Y., Yao, X., Wilson, Z.A., 1050
Qian, Q., and Zhang, D.B. (2009). RETARDED PALEA1 Controls Palea 1051
Development and Floral Zygomorphy in Rice. Plant Physiol 149, 235-244. 1052
Zhang J, Guo D, Chang Y, You C, Li X, Dai X, Weng Q, Zhang J, Chen G, Li X, Liu 1053
H, Han B, Zhang Q, Wu C. (2007). Non-random distribution of T-DNA 1054
insertions at various levels of the genome hierarchy as revealed by analyzing 1055
13,804 T-DNA flanking sequences from an enhancer-trap mutant library. Plant J, 1056
49, 947-959 1057
Zhang, T., Li, Y., Ma, L., Sang, X., Ling, Y., Wang, Y., Yu, P., Zhuang, H., Huang, J., 1058
Wang, N., Zhao, F., Zhang, C., Yang, Z., Fang, L., and He, G. (2017). 1059
LATERAL FLORET 1 induced the three-florets spikelet in rice. Proceedings of 1060
the National Academy of Sciences of the United States of America 114, 9984-1061
9989. 1062
Zheng, M., Y. Wang, Y. Wang, C. Wang, Y. Ren, J. Lv, C. Peng, T. Wu, K. Liu, S. 1063
Zhao, X. Liu, X. Guo, L. Jiang, W. Terzaghi and J. Wan. (2015). DEFORMED 1064
FLORAL ORGAN1 (DFO1) regulates floral organ identity by epigenetically 1065
repressing the expression of OsMADS58 in rice (Oryza sativa). New Phytol 1066
206(4), 1476-1490. 1067
1068
Figure legends 1069
Figure 1. Phenotypes of Spikelets in the Wild Type and nsg1-1 Mutant. 1070
(A) Spikelets of the wild type. A1–A2, A wild-type spikelet; the lemma was removed in 1071
(A2). A3–A7, A wild type spikelet; the lemma was removed in (A3). A4 to A7 show the 1072
surface characters of rg, sl, bp, lo and mrp, respectively. A8, Transverse sections of a 1073
wild-type spikelet showing a similar phenotype to those in (A1–A7). 1074
(B–E) Spikelets of the nsg1-1 mutant. B1–B2, A nsg1-1 spikelet; the lemma was removed 1075
in (B2). B3–B7, A nsg1-1 spikelet; the lemma was removed in (B3). B4 to B7 show 1076
surface characters of the elongated rg, elongated sl, lemma-like sl, and lemma-like mrp, 1077
respectively. B8, Transverse sections of a nsg1-1 spikelet showing a similar phenotype to 1078
those in (B1–B7). 1079
35
C1–C2, A nsg1-1 spikelet; the lemma and the lemma-like palea were removed in (C2). 1080
C3–C5, A nsg1-1 spikelet; the lemma and the lemma-like palea were removed in (C3). 1081
C4 and C5 show the surface characters of the elongated lodicule and lodicule-like 1082
stamen, respectively. C6, Transverse sections of a nsg1-1 spikelet showing a similar 1083
phenotype to those in (C1–C6). 1084
D1–D2, A nsg1-1 spikelet; the additional lemma was separated in (D2). D3–D6, A nsg1-1 1085
spikelet; D4 to D6 show the surface characters of the elongated rg, osl, and isl, 1086
respectively. D7, Transverse sections of a nsg1-1 spikelet showing a similar phenotype to 1087
those in (D1–D6). 1088
E1–E2, A nsg1-1 spikelet; the lemma and the additional lemma were removed in (E2). 1089
E3–E6, A nsg1-1 spikelet. E4 to E6 show the surface characters of the lemma-like rg, osl 1090
and isl, respectively. E7, Transverse sections of a nsg1-1 spikelet showing a similar 1091
phenotype to those in (E1–E6). Red arrowheads indicate the floral organs in the 1092
additional floret. 1093
(F) Schematic diagrams of wild type (WT) and nsg1-1 spikelet structure in transverse1094
section. 1095
(G) Percentage of elongated or lemma-like organs in nsg1-1 spikelets.1096
White type indicates the normal organs, and red type indicates the abnormal organs. 1097
Bars = 1,000 μm in *1, *2 and *3; 500 μm in A8, B8, C6, and D7; 50 μm in all other 1098
images. 1099
1100
Figure 2. Histological and RT-qPCR Analysis of Lateral Organs in Spikelets of the Wild 1101
Type and nsg1-1 Mutant. 1102
(A) Transverse sections of lateral organs in a wild-type spikelet.1103
(B–D) Transverse sections of lateral organs in nsg1-1 spikelets. 1104
A1 shows the rg of the wild type while B1, C1, and D1 show mrp-like rg in nsg1-1 spikelets. 1105
A2 shows the osl of the wild type; B2, C2, and D2 show mrp-like and/or lemma-like osl in 1106
nsg1-1 spikelets. 1107
36
A3 shows the isl of the wild type; B3, C3, and D3 show mrp-like and/or lemma-like isl in 1108
nsg1-1 spikelets. 1109
A4 shows the mrp of the wild type; B4, C4, and D4 show lemma-like mrp in nsg1-1 1110
spikelets. 1111
A5 shows the lodicule and stamen of the wild type; B5, C5, and D5 show abnormal lodicule, 1112
mrp-like lodicule, and mrp-like filament in nsg1-1 spikelets, respectively. 1113
(E–G) RT-qPCR analysis of MFO1, DL, and LHS1 expression. ACTIN was used as a 1114
control. RNA was isolated from flower organs of wild-type and nsg1-1 spikelets. Error bars 1115
indicate SD. At least three replicates were performed, from which the mean value was used 1116
to represent the expression level. 1117
ele, additional lemma; fi, filament; irg, inner rudimentary glume; isl, inner sterile lemma; 1118
le, lemma; lo, lodicule; mrp, marginal region of palea; osl, outer sterile lemma; pa, palea; 1119
st, stamen. 1120
White type indicates the normal organs, and red type indicates the abnormal organs. 1121
Bars = 150 μm. 1122
1123
Figure 3. Scanning Electron Micrographs of Spikelets of the nsg1-1 Mutant at Early 1124
Developmental Stages. 1125
(A) Wild-type spikelets.1126
(B) nsg1-1 spikelets with relatively normal palea primordia.1127
(C) nsg1-1 spikelets with additional lemma or lemma-like palea primordia.1128
A1/B1/C1/, A2/B2/C2, A3/B3/C3, and A4/B4/C4 show spikelets at the Sp4, Sp5, Sp6, 1129
and Sp8 developmental stages, respectively. 1130
ele, additional lemma; elo:elongated lodicule; irg, inner rudimentary glume; isl, inner 1131
sterile lemma; le, lemma; osl, outer sterile lemma; org, outer rudimentary glume; pa, 1132
palea.White type indicates the normal organs, and red type indicates the abnormal organs. 1133
Bars = 100 μm. 1134
1135
Figure 4. Map-based Cloning of NSG1. 1136
37
(A) Schematic illustration of the genomic structure of NSG1. The sites of the mutation in 1137
the nsg1-1, nsg1-2, and nsg1-3 mutants are shown. 1138
(B) Structure of the NSG:GFP fusion complementary vector and the phenotypes of three1139
independent transgenic lines. 1140
(C) Allelism test between the nsg1-1 and nsg1-2 mutants. Three primer pairs were used to1141
detect the distinct sites of the nsg1-1 and/or nsg1-2 mutation. Bars = 1,000 μm. 1142
1143
Figure 5. NSG1 Encodes a Zinc Finger protein with a Single C2H2 Motif.1144
(A) Phylogenetic tree for NSG1-like proteins. The phylogenetic tree was constructed using1145
the neighbor-joining method based on the Jones–Taylor–Thornton matrix-based model. 1146
Bootstrap support values calculated from 1000 replicates are given at the branch nodes. Ac, 1147
Ananas comosus; Aet, Aegilops tauschii; Bd, Brachypodium distachyon; Ca, Capsicum 1148
annuum; Cc, Cajanus cajan; Cia, Cicer arietinum; Do, Dichanthelium oligosanthes; Eg, 1149
Elaeis guineensis; Gh, Gossypium hirsutum; Gm, Glycine max; Jc, Jatropha curcas; Ma, 1150
Musa acuminata; Nt, Nicotiana tabacum; Os, Oryza sativa; Pt, Populus trichocarpa; Sb, 1151
Sorghum bicolor; Si, Setaria italica; Sl, Solanum lycopersicum; Tc, Theobroma cacao; Vr, 1152
Vigna radiata; Vv, Vitis vinifera; Zm, Zea mays.1153
(B) Analysis of the subcellular localization of the NSG1 protein. Bars = 50 µm.1154
(C-E) Analysis of the transcriptional activation of NSG1 using the dual luciferase reporter 1155
assay system. Transactivation activity in rice protoplasts transfected with a pUAS-fLUC 1156
reporter construct, effector constructs fused with GAL4BD, and a p35S-rLUC 1157
normalization construct. (C) Schematic representation of effectors of GAL4BD plus the 1158
full-length or various truncated visions of NSG1. (D) and (E) Measurement of relative 1159
luciferase activity in rice transient assay. VP16, a transcriptional activator, was used as a 1160
positive control and GAL4-BD was regarded as a negative control. Bars and asterisks 1161
represent SD and significant difference at P < 0.01, Student’s t test. 1162
1163
38
Figure 6. Expression Pattern of NSG1. 1164
GFP signal indicating NSG1 expression in transgenic complementation plants harboring 1165
the construct NSG1P:NSG1:GFP. 1166
(A) A whole young panicle (~1 cm length).1167
(B) Several spikelets at developmental stages Sp4 to Sp7.1168
(C) A spikelet at Sp6.1169
(D) A spikelet at Sp7.1170
(E) A primary branch of a panicle (~3.0 cm length).1171
(F) Several spikelets at developmental stages Sp8 and post-Sp8.1172
(G) A spikelet at Sp8.1173
(H) A spikelet at post-Sp8.1174
Bars = 50 μm. 1175
1176
Figure 7. Expression Pattern of LHS1 in Spikelets of the Wild type and the nsg1-1 1177
Mutant. 1178
A–D, I and M show expression of LHS1 in wild type spikelets, using in situ 1179
hybridization.1180
E-H, J-L and N-P show expression of LHS1 in nsg1-1 spikelets, using in situ1181
hybridization.1182
A and E, B and F, C and G, D and H show longitudinal sections of spikelets at stages Sp5 1183
to Sp8, respectively. I –L and M-P show transverse sections of basal region and middle 1184
region of spikelets at stages of Sp8, respectively.1185
irg, inner rudimentary glume; isl, inner sterile lemma; le, lemma; mrp, marginal region of 1186
palea; osl, outer sterile lemma; pa, palea; sl, sterile lemma.1187
Black type indicates the normal expression of genes, and red type indicates the ectopic or 1188
abnormal expression signal of genes. 1189
Bars = 50 μm.1190
39
1191
Figure 8. Direct Regulation of LHS1 and DL Expression by NSG1. 1192
(A) Distribution of potential binding sites in the promoter and ORF regions of LHS1. 1193
Blue bars indicate the DNA fragments amplified in the ChIP assays. Red bars indicate the 1194
DBS-like motifs in the promoter of LHS1. 1195
(B) ChIP-qPCR for P1 site of LHS1 with anti-GFP antibody. ChIP enrichment compared 1196
with the input sample was tested by qPCR. Error bars indicate SD of three repeats. 1197
(C) NSG1WT represses LHS1 expression in vivo. N. benthamiana leaves were 1198
transformed with p35Sm:LUC, p35Sm:LUC plus 35S:NSGWT, pLHS1P-35Sm:LUC, 1199
pLHS1P-35Sm:LUC plus 35S:NSGWT, or pLHS1P-35Sm:LUC plus 35S:NSGnsg1-1. Error 1200
bars indicate SD of three repeats. 1201
(D) NSG1 interacts with OsTPR1, OsTPR2 and OsTPR3 in yeast cells by a Y2H assay. 1202
(E) NSG1 interacts with OsTPR1, OsTPR2 and OsTPR3 in the nucleus of N. 1203
benthamiana leaf cells by a BiFC assay. 1204
(F) ChIP-qPCR for several sites of LHS1 with anti-H3K9ac antibody between WT 1205
panicles and nsg1-1 panicles. Error bars indicate SD of three repeats. 1206
(G) Model of NSG1 repressing the expression of LHS1 by recruiting TPRs-HDACs. 1207
1208
Figure 9. Spikelet Phenotypes of the lhs1 Single Mutants and the nsg1-1 lhs1-z Double 1209
Mutant. 1210
(A) Spikelets of the lhs1-z mutant. A1–A2, lhs1-z spikelet; the lemma and palea were 1211
removed in (A2). A3–A5, lhs1-z spikelet; the lemma and palea were removed in (A3). A4 1212
and A5 show the surface characteristics of rg and sl, respectively. A6–A8, Transverse 1213
sections of lhs1-z spikelet; A7 and A8 show the anatomical structure of osl and isl, 1214
respectively. 1215
(B) Spikelets of the nsg1-1 lhs1-z mutant. B1–B2, nsg1-1 lhs1-z spikelet; the lemma and 1216
palea were removed in (B2). B3–B6, nsg1-1 lhs1-z spikelet. B4 to B6 show surface 1217
characters of the elongated irg, osl and isl, respectively. B7–B9, Transverse sections of 1218
40
nsg1-1 lhs1-z spikelet; B8 and B9 show the anatomical structure of irg, osl and isl, 1219
respectively. 1220
(C) Percentage of elongated or lemma-like organs in nsg1-1 spikelets.1221
(D–F) qPCR analysis of LHS1, DL, and MFO1 expression in spikelets of nsg1-1, lhs1-z, 1222
nsg1-1 lhs1-z double mutant. ACTIN was used as a control. RNA was isolated from rg 1223
and sl of nsg1-1, lhs1-z, nsg1-1 lhs1-z spikelets. Error bars indicate SD. At least three 1224
replicates were performed, from which the mean value was used to represent the 1225
expression level. 1226
irg, inner rudimentary glume; isl, inner sterile lemma; org, outer rudimentary glume; osl, 1227
outer sterile lemma; rg, rudimentary glume; sl, sterile lemma. 1228
Bars = 1,000 μm in *1, *2 and *3; 100 μm in *4, *5, B6; 100 μm in the other images. 1229
1230
Figure 10. Roles of NSG1 in the Specification of Organ Identity in the Rice Spikelet. 1231
(A) In the wild-type spikelet, G1, LHS1, DL, and MFO1 play pivotal roles in1232
specification of lateral organ identities (G1 for rg and sl; LHS1 for le; DL for le; and 1233
MFO1 for mrp and lo). NSG1 acts as a repressor to regulate the specification of lateral 1234
organs, including rg, sl, mrp, lo, and st, by directly repressing LHS1 and DL, as well as 1235
MFO1, and indirectly activating G1 through LHS1. (1) indicates a study that suggests 1236
LHS1 represses G1 (Wang et al., 2017). 1237
(B) In the nsg1 spikelet, with the loss of NSG1 function, LHS1, DL, and MFO1 were1238
ectopically expressed in the rg, sl, mrp, lo, or st, and G1 expression was under-regulated 1239
in the rg and sl, which led to transformation of other lateral organs into lemma-like 1240
organs. In the nsg1-1 lhs1-z mutant, the lemma-like identity was lost completely in the 1241
nsg1-1 sterile lemmas and rudimentary glumes, suggesting that ectopic LHS1 activation 1242
was a key cause of ectopic formation of lemma-like tissue in these organs. The gene 1243
symbols in green type show a normal expression domain, those in red type show an 1244
41
ectopic expression domain, and those in grey type show an under-regulation expression 1245
domain. 1246
fi, filament; irg, inner rudimentary glume; isl, inner sterile lemma; le, lemma; le-like isl, 1247
lemma like inner rudimentary glume; le-like mrp, lemma like marginal region of palea; 1248
le/mrp-like fi, lemma and/or marginal region of palea like filament; le/mrp like lo, lemma 1249
and/or marginal region of palea like lodicule; le/mrp-like osl, lemma and/or marginal 1250
region of palea like outer sterile lemma; lo, lodicule; mrp-like irg, marginal region of 1251
palea like inner rudimentary glume; mrp, marginal region of palea; osl, outer sterile 1252
lemma. 1253
1254
Figure 1A1 A2 A3 A4 A5 A8
B1 B2 B3 B4 B5 B8
C1 C2 C3 C4 C6
D1 D2 D3 D4 D5 D7
B6
B7
A6 A7
C5
D6
E1 E2 E3 E4 E6E5 E7
org
irgosl
isllo
bpmrp
le pa
bp mrplo
rg sl
st
pi
st
osl isl
le
bp
mrplo
pi
le pamrp
bp
lo
osl
isl
irg
irg osl isl
lo mrp
irgosl
isl
le
mrp
mrp
bp
lo
lofi
fipi
fi
le pa lo lo fi lo
fi
irg
osl
irg
oslisl
le
mrp
mrp
bp
lo
lo
fi
fipifi
oslisl
ele
lepa lo
lo
pi fi
islosl
elele
bp lo
lo
mrp
fi
mrplo
ele le ele lepa
osl
isl
irg
osl
isl
irg
ele lepa
irg osl isl
papa
F G
st
lo
papi
0102030405060708090
100
org irg osl isl pa lo st
Pre
cent
age(
%)
absence of orgelongation/wideninglemma-likenormal
WT nsg1-1
Figure 1. Phenotypes of Spikelets in the Wild Type and nsg1-1 Mutant.(A) Spikelets of the wild type. A1–A2, A wild type spikelet; the lemma was removed in (A2). A3–A7, A wild-typespikelet; the lemma was removed in (A3). A4 to A7 show the surface characters of rg, sl, bp, lo and mrp, respectively.A8, Transverse sections of a wild-type spikelet showing a similar phenotype to those in (A1–A7).(B–E) Spikelets of the nsg1-1 mutant. B1–B2, A nsg1-1 spikelet; the lemma was removed in (B2). B3–B7, A nsg1-1spikelet; the lemma was removed in (B3). B4 to B7 show surface characters of the elongated rg, elongated sl, lemma-likesl, and lemma-like mrp, respectively. B8, Transverse sections of a nsg1-1 spikelet showing a similar phenotype to thosein (B1–B7).C1–C2, A nsg1-1 spikelet; the lemma and the lemma-like palea were removed in (C2). C3–C5, A nsg1-1 spikelet; thelemma and the lemma-like palea were removed in (C3). C4 and C5 show the surface characters of the elongated lodiculeand lodicule-like stamen, respectively. C6, Transverse sections of a nsg1-1 spikelet showing a similar phenotype to thosein (C1–C6).D1–D2, A nsg1-1 spikelet; the additional lemma was separated in (D2). D3–D6, A nsg1-1 spikelet; D4 to D6 show thesurface characters of the elongated rg, osl, and isl, respectively. D7, Transverse sections of a nsg1-1 spikelet showing asimilar phenotype to those in (D1–D6).E1–E2, A nsg1-1 spikelet; the lemma and the additional lemma were removed in (E2). E3–E6, A nsg1-1 spikelet. E4 toE6 show the surface characters of the lemma-like rg, osl and isl, respectively. E7, Transverse sections of a nsg1-1spikelet showing a similar phenotype to those in (E1–E6). Red arrowheads indicate a fused organ of lodicule and palea inan incomplete extra floret.(F) Schematic diagrams of wild type (WT) and nsg1-1 spikelet structure in transverse section.(G) Percentage of elongated or lemma-like organs in nsg1-1 spikelets.absence of org, absence of outer rudimentary glume; bp, body of palea; ele, additional lemma; fi, filament; irg, innerrudimentary glume; isl: inner sterile lemma; le, lemma; le-like isl, lemma like inner rudimentary glume; le-like mrp,lemma like marginal region of palea; le/mrp-like fi, lemma and/or marginal region of palea like filament; le/mrp-like irg:lemma and/or marginal region of palea like inner rudimentayu glume; le/mrp like lo: lemma and/or marginal region ofpalea like lodicule; le/mrp-like osl: lemma and/or marginal region of palea like outer sterile lemma; lo: lodicule; lo-likefi: lodicule like filament; mrp: marginal region of palea; org, outer rudimentary glume; osl: outer sterile lemma; pa:palea; pi: pistil; st: stamen.White type indicates the normal organs, and red type indicates the abnormal organs.Bars = 1,000 μm in *1, *2 and *3; 500 μm in A8, B8, C6, and D7; 50 μm in all other images.
Figure 2A2 A4 A5
B1 B2 B3 B5
A3
B4
C1 C2C3 C5C4
D1 D2
D3
D5
D4
A1
0
1
2
3
4
5
6
7
Rel
ativ
e ex
pres
sion
LHS1
J10
nsg1-1
0
5
10
15
20
25
Rel
ativ
e ex
pres
sion
DLJ10
nsg1-1
0
1
2
3
4
5
6
Rel
ativ
e ex
pres
sion
MFO1
J10
nsg1-1
E F G
mrplo
fi
mrplo
fi
mrp
mrp
lo
Figure 2. Histological and qPCR Analysis of Lateral Organs in Spikelets of the Wild Type and nsg1-1 Mutant.(A) Transverse sections of lateral organs in a wild-type spikelet.(B–D) Transverse sections of lateral organs in nsg1-1 spikelets.A1 shows the rg of the wild type while B1, C1, and D1 show mrp-like rg in nsg1-1 spikelets.A2 shows the osl of the wild type; B2, C2, and D2 show mrp-like and/or lemma-like osl in nsg1-1 spikelets.A3 shows the isl of the wild type; B3, C3, and D3 show mrp-like and/or lemma-like isl in nsg1-1 spikelets.A4 shows the mrp of the wild type; B4, C4, and D4 show lemma-like mrp in nsg1-1 spikelets.A5 shows the lodicule and stamen of the wild type; B5, C5, and D5 show abnormal lodicule, mrp-like lodicule, and mrp-like filament in nsg1-1 spikelets, respectively.(E–G) qPCR analysis of MFO1, DL, and LHS1 expression. ACTIN was used as a control. RNA was isolated fromflower organs of wild type and nsg1-1 spikelets . Error bars indicate SD. At least three replicates were performed, fromwhich the mean value was used to represent the expression level.ele, additional lemma; fi, filament; irg, inner rudimentary glume; isl, inner sterile lemma; le, lemma; lo, lodicule; mrp,marginal region of palea; osl, outer sterile lemma; pa, palea; st, stamen.White type indicates the normal organs, and red type indicates the abnormal organs.Bars = 150 μm.
Figure 3
orgirg
le
pa
irgosl
pale
orgirg
osl isl
le
pa
irg
osl isl
le pa
elo
irg
isl
le pa
irg
osl
isl
islele
lepa
elo
irgosl
isl
lepaelo
elo
irg
lepa
eirg
leele le
le le lepa
papa
st
A1 A2 A3 A4
B1 B2 B3 B4
C1 C2 C3 C4
Figure 3. Scanning Electron Micrographs of Spikelets of the nsg1-1 Mutant at Early Developmental Stages. (A) Wild-type spikelets.(B) nsg1-1 spikelets with relatively normal palea primordia.(C) nsg1-1 spikelets with additional lemma or lemma-like palea primordia.A1/B1/C1/, A2/B2/C2, A3/B3/C3, and A4/B4/C4 show spikelets at the Sp4, Sp5, Sp6, and Sp8 developmental stages,respectively.ele, additional lemma; elo:elongated lodicule; irg, inner rudimentary glume; isl, inner sterile lemma; le, lemma; osl,outer sterile lemma; org, outer rudimentary glume; pa, palea.White type indicates the normal organs, and red type indicates the abnormal organs.Bars = 100 μm.
Figure 4
A
B
C
nsg1-1NSG:GFPcom-1
nsg1-1NSG:GFPcom-3
nsg1-1NSG:GFPcom-7
comF2 comR1
NSG GFP NOS
nsg1-1 nsg1-2 F1
T-DNA insertion (-1202bp)
LB3 RBF2 1R F4 OER
nsg1-2 nsg1-3 nsg1-1
RMD_ITL-04Z11DF46Transversion
A-T (+124bp/42aa)Insertion(+444bp/147aa)
TGCAGGGTGGTTA
OEF
Figure 4. Map-based cloning of NSG1. (A) Schematic illustration of the genomic structure of NSG1. The sites of the mutation in the nsg1-1, nsg1-2, andnsg1-3 mutants are shown.(B) Structure of the NSG:GFP fusion complementary vector and the phenotypes of three independent transgenic lines.(C) Allelic test between the nsg1-1 and nsg1-2 mutants. Three primer pairs were used to detect the distinct site of thensg1-1 and/or nsg1-2 mutation.Bars = 1,000 μm.
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
Rel
ativ
e lu
cife
rase
act
ivity
DoOEL25293 ZmXP008669354
SbKXG26384 ZmLOC100383297 SiXP004975723
NSG1 BdKQJ82820
AetEMT04360 Os02g35460
MaXP009381578 EgXP010913843
AcOAY79850 ZmXP 008652751
Os09g26210 BdLOC100838938
TcEOY29371 JcXP012076458
At5g10970 At5g25160
At1g80730 PtXP002324478
VrXP014508495 GmXP003550661 CcKYP57451
CiaXP004508237 GhXP016698842
VvXP010656831 SlNP001315357
NtXP009614282
99
93
96
99
85
82
60
89
58
76
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Figure 5. NSG1 Encodes a Zinc Finger protein with a Single C2H2 Motif.(A) Phylogenetic tree for NSG1-like proteins. The phylogenetic tree was constructed using the neighbor-joining methodbased on the Jones–Taylor–Thornton matrix-based model. Bootstrap support values calculated from 1000 replicates aregiven at the branch nodes. Ac, Ananas comosus; Aet, Aegilops tauschii; Bd, Brachypodium distachyon; Ca, Capsicumannuum; Cc, Cajanus cajan; Cia, Cicer arietinum; Do, Dichanthelium oligosanthes; Eg, Elaeis guineensis; Gh,Gossypium hirsutum; Gm, Glycine max; Jc, Jatropha curcas; Ma, Musa acuminata; Nt, Nicotiana tabacum; Os, Oryzasativa; Pt, Populus trichocarpa; Sb, Sorghum bicolor; Si, Setaria italica; Sl, Solanum lycopersicum; Tc, Theobromacacao; Vr, Vigna radiata; Vv, Vitis vinifera; Zm, Zea mays.(B) Analysis of the subcellular localization of the NSG1 protein. Bars = 50 µm.(C-E) Analysis of the transcriptional activation of NSG1 using the dual luciferase reporter assay system. Transactivationactivity in rice protoplasts transfected with a pUAS-fLUC reporter construct, effector constructs fused with GAL4BD,and a p35S-rLUC normalization construct. (C) Schematic representation of effectors of GAL4BD plus the full-length orvarious truncated visions of NSG1. (D) and (E) Measurement of relative luciferase activity in rice transient assay.VP16, a transcriptional activator, was used as a positive control and GAL4-BD was regarded as a negative control. Barsand asterisks represent SD and significant difference at P < 0.01, Student’s t test.
Figure 6
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Figure 6. Expression Pattern of NSG1. GFP signal indicating NSG1 expression in transgenic complementary plants harboring the construct NSGP1:NSG1:GFP. (A) A whole young panicle (~1 cm length).(B) Several spikelets at developmental stages Sp4 to Sp7.(C) A spikelet at Sp6.(D) A spikelet at Sp7.(E) A primary branch of a panicle (~3.0 cm length).(F) Several spikelets at developmental stages Sp8 and post-Sp8.(G) A spikelet at Sp8.(H) A spikelet at post-Sp8.Bars = 50 μm.
Figure7
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Figure 7. Expression Pattern of LHS1 gene in Spikelets of the Wild type and the nsg1-1 Mutant.A–D, I and M show expression of LHS1 in wild type spikelets, using in situ hybridization.E-H, J-L and N-P show expression of LHS1 in nsg1-1 spikelets, using in situ hybridization.A and E, B and F, C and G, D and H show longitudinal sections of spikelets at stages Sp5 to Sp8, respectively.I –L and M-P show transverse sections of basal region and middle region of spikelets at stages of Sp8, respectively.irg, inner rudimentary glume; isl, inner sterile lemma; le, lemma; mrp, marginal region of palea; osl, outer sterilelemma; pa, palea; sl, sterile lemma.Black type indicates the normal expression of genes, and red type indicates the ectopic or abnormal expression signalof genes. Bars = 50 μm.
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Figure 8. Direct Regulation of LHS1 Expression by NSG1.(A) Distribution of potential binding sites in the promoter and ORF regions of LHS1. Blue bars indicate the DNAfragments amplified in the ChIP assays. Red bars indicate the DBS-like motifs in the promoter of LHS1.(B) ChIP-qPCR for P1 site of LHS1 with anti-GFP antibody. ChIP enrichment compared with the input samplewas tested by qPCR. Error bars indicate SD of three repeats.(C) NSG1WT represses LHS1 expression in vivo. Tobacco leaves were transformed with p35Sm:LUC,p35Sm:LUC plus 35S:NSGWT, pLHS1P-35Sm:LUC, pLHS1P-35Sm:LUC plus 35S:NSGWT, or pLHS1P-35Sm:LUC plus 35S:NSGnsg1-1. Error bars indicate SD of three repeats.(D) NSG1 interacts with OsTPR1, OsTPR2 and OsTPR3 in yeast cells by a Y2H assay .(E) NSG1 interacts with OsTPR1, OsTPR2 and OsTPR3 in the nuclear of tobacco leave cells by a BiFC assay.(F) ChIP-qPCR for several sites of LHS1 with anti-H3K9ac antibody between WT panicles and nsg1-1 panicles.Error bars indicate SD of three repeats.(G) Model of NSG1 repressing the expression of LHS1 by recruiting TPRs-HDACs.
Figure 9
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Figure 9. Spikelet Phenotypes of the lhs1-z Single Mutants and the nsg1-1+lhs1-z Double Mutant.(A) Spikelets of the lhs1-z mutant. A1–A2, lhs1-z spikelet; the lemma and palea were removed in (A2). A3–A5,lhs1-z spikelet; the lemma and palea were removed in (A3). A4 and A5 show the surface characters of rg and sl,respectively. A6–A8, Transverse sections of lhs1-z spikelet; A7 and A8 show the anatomical structure of osl and isl,respectively.(B) Spikelets of the nsg1-1+lhs1-z mutant. B1–B2, nsg1-1+lhs1-z spikelet; the lemma and palea were removed in(B2).B3–B6, nsg1-1+lhs1-z spikelet. B4 to B6 show surface characters of the elongated irg, osl and isl, respectively. B7–B9, Transverse sections of nsg1-1+lhs1-z spikelet; B8 and B9 show the anatomical structure of irg, osl and isl,respectively.(C) Percentage of elongated or lemma-like organs in nsg1-1 spikelets.(D–F) qPCR analysis of LHS1, DL, and MFO1 expression in spikelets of nsg1-1, lhs1-z, nsg1-1+lhs1-z doublemutant. ACTIN was used as a control. RNA was isolated from rg and sl of nsg1-1, lhs1-z, nsg1-1+lhs1-z spikelets.Error bars indicate SD. At least three replicates were performed, from which the mean value was used to representthe expression level.irg, inner rudimentary glume; isl, inner sterile lemma; org, outer rudimentary glume; osl, outer sterile lemma; rg,rudimentary glume; sl, sterile lemma.Bars = 1,000 μm in *1, *2 and *3; 100 μm in *4, *5, B6; 100 μm in the other images.
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Figure 10. Roles of NSG1 in the Specification of Organ Identity in the Rice Spikelet.(A) In the wild type spikelet, G1, LHS1, DL, and MFO1 play pivotal roles in specification of lateral organ identities (G1for rg and sl; LHS1 for le; DL for le; and MFO1 for mrp and lo). NSG1 acts as a repressor to regulate the specificationof lateral organs, including rg, sl, mrp, lo, and st, by directly repressing LHS1 and DL, as well as MFO1, and indirectlyactivating G1 through LHS1. (1) indicates a study that suggests LHS1 represses G1 (Wang et al., 2017).(B) In the nsg1 spikelet, with the loss-of-function of NSG1, LHS1, DL, and MFO1 were ectopically expressed in the rg,sl, mrp, lo, or st, and G1 expression was under-regulated in the rg and sl, which led to transformation of other lateralorgans into lemma-like organs. In the nsg1-1+lhs1-z mutant, the lemma-like identity were lost completely in the nsg1-1sterile lemmas and rudimentary glumes, suggesting that ectopic LHS1 activation was an key cause of ectopic formationof lemma-like tissue in these organs. The gene symbols in green type show a normal expression domain, those in redtype show an ectopic expression domain, and those in grey type show an under-regulation expression domain.fi, filament; irg, inner rudimentary glume; isl, inner sterile lemma; le, lemma; le-like isl, lemma like inner rudimentaryglume; le-like mrp, lemma like marginal region of palea; le/mrp-like fi, lemma and/or marginal region of palea likefilament; le/mrp like lo, lemma and/or marginal region of palea like lodicule; le/mrp-like osl, lemma and/or marginalregion of palea like outer sterile lemma; lo, lodicule; mrp-like irg, marginal region of palea like inner rudimentaryglume; mrp, marginal region of palea; osl, outer sterile lemma.
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Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
DOI 10.1105/tpc.19.00682; originally published online December 5, 2019;Plant Cell
Hao Zheng, Jun Tang, Yinghua Ling, Zhenglin Yang, Guang-Hua He and Yun-Feng LiHui zhuang, Hong-Lei Wang, Ting Zhang, Xiao-Qin Zeng, Huan Chen, Zhong-Wei Wang, Jun Zhang,
in RiceNONSTOP GLUMES 1 Encoding a C2H2 Zinc Finger Protein That Regulates Spikelet Development
This information is current as of February 1, 2020
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