Wheat TaSPL8 Modulates Leaf Angle Through Auxin and ...Jun 17, 2019 · 147 that the flag leaf...
Transcript of Wheat TaSPL8 Modulates Leaf Angle Through Auxin and ...Jun 17, 2019 · 147 that the flag leaf...
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Wheat TaSPL8 Modulates Leaf Angle Through Auxin and 1
Brassinosteroid Signaling 2
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Kaiye Liuabc,1, Jie Caoabc,1, Kuohai Yuabc, Xinye Liuabc, Yujiao Gaoabc, Qian Chenabc, 4 Wenjia Zhangabc, Huiru Pengabc, Jinkun Duabc, Mingming Xinabc, Zhaorong Huabc, 5 Weilong Guoabc, Vincenzo Rossid, Zhongfu Niabc, Qixin Sunabc, and Yingyin Yaoabc* 6
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aState Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing, 100193, China 8
bKey Laboratory of Crop Heterosis and Utilization (MOE), China Agricultural University, Beijing, 100193, China 9
cBeijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing, 100193, China 10
dCouncil for Agricultural Research and Economics, Research Centre for Cereal and Industrial Crops, I-24126 11
Bergamo, Italy 12
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1These authors contributed equally to this work. 14
*Corresponding author: Yingyin Yao 15
China Agricultural University, Beijing, 100193, China 16
Tel: 86 +10 62732304 17
Email: [email protected] 18
Running Title: Wheat TaSPL8 affects leaf angle. 19
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One-sentence summary: 21
Wheat TaSPL8 regulates lamina joint development and leaf angle through auxin 22
signaling and the brassinosteroid biosynthesis pathway. 23
Author contributions 24
Z.N., Q.S., and Y.Y. conceived this project and designed all experiments. K.L., J.C, 25
K.Y., X.L., Y.G., M.X., W.Z. and W.G. performed the experiments and data analysis. 26
Q.C., H.P., and Z.H. contributed to the wheat transformation. Y.Y., V.R., and J.D. 27
contributed to manuscript writing and revision, and R.X. analyzed data. 28
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Plant Physiology Preview. Published on June 17, 2019, as DOI:10.1104/pp.19.00248
Copyright 2019 by the American Society of Plant Biologists
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Abstract 30
In grass crops, leaf angle is determined by development of the lamina joint, the tissue 31
connecting the leaf blade and sheath, and is closely related to crop architecture and 32
yield. In this study, we identified a mutant generated by fast neutron radiation that 33
exhibited an erect leaf phenotype caused by defects in lamina joint development. 34
Map-based cloning revealed that the gene TaSPL8, encoding a SQUAMOSA 35
PROMOTER BINDING-LIKE (SPL) protein, is deleted in this mutant. TaSPL8 36
knock-out mutants exhibit erect leaves due to loss of the lamina joint, compact 37
architecture, and increased spike number especially in high planting density, suggesting 38
similarity with its LIGULESS1 (LG1) homologs in maize (Zea mays) and rice (Oryza 39
sativa). Hence, LG1 could be a robust target for plant architecture improvement in grass 40
species. Common wheat (Triticum aestivum, 2n=6×=42; BBAADD) is an 41
allohexaploid containing A/B/D subgenomes and the homoeologous gene of TaSPL8 42
from the D subgenome contributes to the length of the lamina joint to a greater extent 43
than that from the A and B subgenomes. Comparison of the transcriptome between the 44
Taspl8 mutant and the wild type revealed that TaSPL8 is involved in the activation of 45
genes related to auxin and brassinosteroid pathways and cell elongation. TaSPL8 binds 46
to the promoters of the AUXIN RESPONSE FACTOR (TaARF6) gene and of the 47
brassinosteroid biogenesis gene CYP90D2 (TaD2) and activates their expression. 48
These results indicate that TaSPL8 might regulate lamina joint development through 49
auxin signaling and the brassinosteroid biosynthesis pathway. 50
Introduction 51
Leaf angle, defined as the inclination between the leaf blade midrib and the stem, 52
directly influences canopy structure and consequentially affects yield (Mantilla-Perez 53
and Salas Fernandez, 2017). Plants with erect leaves have an increased capacity to 54
intercept light and higher photosynthetic efficiency, which results in improved grain 55 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
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filling (Sinclair et al., 1999). More efficient grain filling enables planting of larger 56
populations with a greater leaf area index (LAI). Hence, using plants with more erect 57
leaves generally improves the yield per unit of cultivated area (Pendleton et al., 1968; 58
Duvick, 2005; Lee and Tollenaar, 2007; Lauer et al., 2012). For example, the maize 59
(Zea mays) liguleless2 (lg2) mutant has erect leaves and produces 40% more grain than 60
its counterpart with horizontal-type leaves due to the relative efficiency of CO2 fixation 61
per unit of incoming sunlight, which increases as the leaf angle decreases (Pendleton et 62
al., 1968). In rice (Oryza sativa), the Osdwarf4-1 mutant exhibits an erect leaf 63
phenotype that is associated with brassinosteroid (BR) deficiency and has enhanced 64
grain yields under dense planting populations (Sakamoto et al., 2006). In wheat 65
(Triticum sp.), the important characteristics of the ideal plant architecture (ideotype) 66
include short and strong stems with few, small, and erect leaves (Donald, 1968). Wheat 67
genotypes with erect leaves also have a superior net carbon fixation capacity during 68
grain filling (Austin et al., 1976). Therefore, breeding grass crops for more erect leaves 69
is a reasonable strategy for improving crop productivity. 70
71
Leaf angle depends on cell division, expansion, and cell wall composition in the lamina 72
joint (including the auricle and ligule), which connects the leaf blade and sheath (Kong 73
et al., 2017; Zhou et al., 2017). Genetic and functional studies indicated that various 74
factors are involved in regulating lamina joint development, and consequentially affect 75
leaf angle. Phytohormones, such as BR and auxin, play crucial roles in regulating the 76
lamina inclination (Luo et al., 2016). In rice, loss of function of BR biosynthetic genes, 77
such as dwarf4-1 (Sakamoto et al., 2006), ebisu dwarf (d2) (Hong et al., 2003), 78
brassinosteroid-deficient dwarf1 (brd1) (Hong et al., 2002), and chromosome segment 79
deleted dwarf 1(csdd1) (Li et al., 2013) results in reduced leaf inclination. Lamina joint 80
development is also associated with BR signaling, as reported by studies of mutants 81
less sensitive to BR, such as the BR-defective mutant d61 (Yamamuro et al., 2000) and 82
transgenic rice plants with suppressed expression of BRI1-Associated receptor Kinase 83 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
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1 (OsBAK1) (Li et al., 2009) and BRASSINAZOLE-RESISTANT1 (OsBZR1) (Bai et al., 84
2007). Similarly, auxin biosynthesis and signaling pathways influence lamina joint 85
development. Both the gain-of-function rice LEAF INCLINATION1 (OsLC1) (Zhao et 86
al., 2013) mutant and plants overexpressing Gretchen Hagen 3 acyl acid amido 87
synthetases (GH3) family members, including OsGH3-1, -2, -5, and -13 (Du et al., 88
2012; Zhao et al., 2013; Zhang et al., 2015), have reduced auxin levels and show an 89
enlarged leaf angle due to stimulated cell elongation at the lamina joint region. Rice 90
transgenic lines overexpressing the auxin response factor OsARF19 exhibit an enlarged 91
lamina inclination related to the increase of adaxial cell division (Zhang et al., 2015). 92
Furthermore, high concentrations of the auxin indole-3-acetic acid (IAA) influence leaf 93
inclination by interacting with BR (Wada et al., 1981; Cao and Chen, 1995) and 94
ethylene, which also participates in BR-induced leaf inclination (Jang et al., 2017). 95
Therefore, crosstalk occurs between different phytohormones in regulating lamina joint 96
development and leaf inclination. 97
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Genetic approaches have identified several genes that affect lamina joint development 99
in rice and maize (Mantilla-Perez and Salas Fernandez, 2017). For instance, rice 100
BRASSINOSTEROID UPREGULATED 1-LIKE1 (OsBUL1), encoding a typical bHLH 101
transcription factor, is preferentially expressed in the lamina joint and affects leaf angle 102
by controlling cell elongation (Jang et al., 2017). Similarly, overexpression or ectopic 103
expression of other bHLH transcription factor genes, such as LAX PANICLE (LAX), 104
OsbHLH153, OsbHLH173, OsbHLH174, BRASSINOSTEROID UPREGULATED1 105
(BU1), INCREASED LAMINA INCLINATION1 (ILI1), and POSITIVE REGULATOR 106
OF GRAIN LENGTH1 (PGL1), results in a larger lamina inclination (Komatsu et al., 107
2003; Tanaka et al., 2009; Zhang et al., 2009; Dong et al., 2018). LEAF 108
INCLINATION2 (LC2), a VIN3-like protein, regulates lamina inclination by 109
repressing adaxial cell division in the collar region (Zhao et al., 2010). An increased 110
leaf angle 1 (ila1) mutant shows increased leaf angle due to abnormal vascular bundle 111 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
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formation and cell wall composition in the lamina joint (Ning et al., 2011). It was 112
suggested that rice SPX1 (SYG1/Pho81/XPR1) could interact with Regulator of Leaf 113
Inclination 1 (RLI1) and negatively regulate leaf inclination by affecting lamina joint 114
cell elongation (Ruan et al., 2018). Mutations in LIGULELESS1 (LG1), which encodes 115
a conserved SPL transcription factor in maize and rice, result in an erect leaf phenotype 116
with a complete loss of the auricle and ligule (Moreno et al., 1997; Lee et al., 2007). A 117
single-nucleotide polymorphism in the upstream region of OsLG1 may be responsible 118
for its expression and affects panicle closure or spreading by controlling cell 119
proliferation at the panicle pulvinus (Ishii et al., 2013; Zhu et al., 2013). However, the 120
contribution of LG1 to potential yield and its mechanism in affecting lamina joint 121
development are still unknown. 122
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In wheat, genetic approaches have identified several quantitative trait loci (QTL) that 124
control leaf inclination. For example, QTL analysis of the markers for flag leaf angle 125
from durum wheat (Triticum durum) populations indicated the presence of large-effect 126
QTL on chromosomes 2A, 2B, 3A, 3B, 4B, 5B, and 7A (Isidro et al., 2012). Several 127
environmentally-stable QTL for flag leaf angle were mapped on chromosomes 1B, 3D, 128
5A, 6B, 7B, and 7D using two recombinant inbred line (RIL) populations (Wu et al., 129
2015; Liu et al., 2018b). However, to date, no genes have been shown to be directly 130
involved in the control of leaf angle in wheat. In this study, we characterized a wheat 131
(Triticum aestivum) mutant with a multi-gene deleted region, which exhibits erect 132
leaves and compact plant architecture. We demonstrate that the TaSPL8 gene, which 133
encodes the SBP domain-containing transcription factor and is homologous to LG1 in 134
maize and rice, is responsible for this mutant phenotype. Our results indicate that 135
TaSPL8 regulates lamina joint development through affecting auxin response and BR 136
biogenesis pathways, thus providing information about the mechanisms linking the 137
activity of SPL family members and hormone-mediated regulation of leaf angle. 138
Results 139 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
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The cpa mutant exhibits defects in lamina joint development 140
To study the molecular mechanism that controls leaf angle in wheat, we screened for 141
mutants with erect leaves from a wheat population mutagenized by fast neutron 142
radiation. We identified a mutant, named cpa for compact plant architecture, with a 143
smaller leaf angle and a more compact plant architecture compared to wheat cultivar 144
Liaochun10 (LC10) (Figure 1A). We measured the flag leaf angle between the midrib 145
of the flag leaf and the internode below the spike in the cpa mutant and LC10 and found 146
that the flag leaf angle was 11.50˚ and 28.23˚ in the cpa mutant and LC10, respectively 147
(Figure 1B and 1C). The smaller flag leaf angle of the cpa mutant is mainly caused by 148
abnormal development of the lamina joint (Figure 1D). In particular, the length of the 149
lamina joint of the flag leaf in the cpa mutant is 1.33 mm, while in LC10 it is 2.64 mm 150
(Figure 1E). Further morphological observation through scanning electron microscopy 151
showed that the lamina joint is much smaller in the cpa mutant than in LC10 due to a 152
decrease in cell number, while no changes in cell size were detected (Figure 1F and 153
1G). These results indicate that the cpa mutant exhibits defects in lamina joint 154
development. 155
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Map-based cloning of mutated gene 157
A genetic linkage analysis of 450 F2 individuals derived from a cross between LC10 158
and the cpa mutant was performed. A total of 335 individual plants showed a large 159
lamina joint like LC10, while 115 plants showed a small lamina joint like the cpa 160
mutant. This result indicated that the lamina joint defect of the cpa mutant is caused by 161
a single recessive gene with a segregation ratio of ~3:1 (χ2 = 0.074 < χ2(0.05,1) = 3.84) of 162
the normal phenotype versus the mutant phenotype. We designated the dominant 163
wild-type gene as Compact plant architecture (TaCPA). 164
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Following a map-based cloning approach using 450 F2 individuals, TaCPA was found 166
to co-segregate with markers 2D-235, 2D1377, LMC-3, and 2D-173 on chromosome 167 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
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2D (Figure 2A). By a larger segregation population consisting of 8352 individuals we 168
narrowed down the candidate region of TaCPA to ~19 Mb between LMC-3 and 2D-173 169
(Figure 2B and 2C). Generation of additional markers in this region allowed the 170
mapping of TaCPA as being completely linked to LMC-15, -18, -19, -23, -60, -71, and 171
-98 (Figure 2B and 2C). Near-isogenic lines (NILs) in the cpa genetic background 172
containing the LC10 allele (NILLC10) and NILs in the LC10 genetic background 173
containing the cpa allele (NILcpa) were generated through backcrossing of 174
heterozygous F1 plants to LC10 and the cpa mutant for three generations (BC3), 175
respectively (Supplemental Figure S1). The backcrossed line containing a 176
heterozygous fragment from markers LMC-3 and 2D-173 was selected. We found that 177
in NILLC10 plants, the flag leaf angle and lamina joint length is significantly increased 178
compared to the cpa mutant (Figure 2D), while these parameters are decreased in the 179
NILcpa plants compared to LC10 (Figure 2E). This confirmed that TaCPA is located in 180
the interval we have identified. 181
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The region between markers LMC-3 and 2D-173 contains a total of 257 predicted genes 183
in the wheat Chinese Spring inbred line genome IWGSCv1(Appels et al., 2018). To 184
map TaCPA at a higher resolution, we performed bulked segregant RNA sequencing 185
(BSR-Seq), which makes use of the quantitative reads of RNA-Seq to efficiently map 186
genes (Liu et al., 2012). RNA samples were extracted from 30 homozygous F2-derived 187
F3 lines with normal or mutant phenotypes and from cpa and LC10 plants and were 188
combined into four separate pools used for RNA-Seq. Reads located in the region 189
between markers LMC-3 and 2D-173 were analyzed and the results indicated that 83 190
genes, distributed within this region, were expressed in both LC10 and homozygous 191
normal phenotype pools, but were absent in the cpa mutant and mutant phenotype 192
pools. These 83 genes could have been deleted in the cpa mutant by fast neutron 193
radiation. To test this hypothesis, 10 genes located within and 2 genes located outside 194
of the region (LMC-3 to 2D-173) were randomly selected and subjected to PCR 195 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
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amplification using genomic DNA of cpa and LC10 plants (Supplemental Figure S2A). 196
We found that all 10 genes within the LMC-3-2D-173 region are absent in the cpa 197
genome, while the 2 external genes are present (Supplemental Figure S2B). The 198
specificity of the primers used for the experiments was further assessed using Chinese 199
Spring null-tetrasomic lines. PCR products were not obtained in the lines with a 200
chromosome 2D deletion, but were present in the other null-tetrasomic lines 201
(Supplemental Figure S2B). These results indicate that the chromosome fragment 202
containing TaCPA is deleted in the cpa mutant. 203
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TaSPL8, the homolog of LG1 in maize and rice, affects lamina joint development 205
and plant architecture in wheat. 206
Among the 83 genes deleted in the cpa mutant, one gene (TraesCS2D01G502900), 207
containing three exons and two introns, encodes a plant-specific SBP 208
domain-containing transcription factor, SPL, with a total protein length of 407 amino 209
acids. TraesCS2D01G502900 shows high amino acid similarity to OsLG1(SPL8) in 210
rice, ZmLG1 in maize, and SbLG1 in sorghum (Sorghum bicolor) (Figure 3A), which 211
are involved in controlling auricle, ligule, and lamina joint development (Moreno et al., 212
1997; Lee et al., 2007). Therefore, we renamed TaCPA as TaSPL8, a candidate gene for 213
lamina joint development in wheat. The expression of TaSPL8 in various wheat tissues 214
were detected by RT-qPCR, and we found that the TaSPL8 transcript is highly 215
expressed in the lamina joint and young spikes (Figure 3B). 216
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Wheat is a hexaploid species that possesses three subgenomes originating from the 218
fusion of genomes from three diploid ancestral species (Triticum urartu, Aegilops 219
speltoides, and Aegilops tauschii) (El Baidouri et al., 2017). Accordingly, three 220
TaSPL8 homoeologs were identified from the wheat genome sequences database 221
(IWGSCv1, (Appels et al., 2018)) and designated TaSPL8-2A, TaSPL8-2B, and 222
TaSPL8-2D, respectively. To determine whether the loss of TaSPL8-2D is responsible 223 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
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for the cpa mutant phenotype, we knocked out this gene using the clustered regularly 224
interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) 225
system. Due to the nucleotide similarity among TaSPL8-2A/2B/2D, single-guide RNA 226
(sgRNA) specifically targeting to TaSPL8-2D cannot be designed. Thus, a sgRNA 227
targeting a conserved region within the first exon of the TaSPL8-2A, TaSPL8-2B, and 228
TaSPL8-2D was designed and the CRISPR/Cas9 vector pBUE411::sgRNA was 229
introduced into LC10 (Figure 3C). A total of 10 independent transgenic T0 plants (KO 230
lines) were obtained and 3 lines with all three homoeologous genes mutated 231
simultaneously were selected for further analysis. Frameshift mutations in all three 232
subgenomes were also detected in the T1 offspring (Figure 3D). The KO-2-1 line was 233
backcrossed to LC10 to generate the TaSPL8-2A/2B/2d single mutant, which showed a 234
smaller lamina joint and flag leaf angle similar to the cpa mutant, compared with LC10 235
(Figure 3E and 3F). These results indicated that the loss of TaSPL8-2D is responsible 236
for the cpa mutant phenotype. 237
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Three homoeologous genes of TaSPL8 differentially contribute to lamina joint 239
growth 240
One extraordinary feature of polyploid evolution is genomic asymmetry, which is the 241
predominant control of a variety of morphological, physiological, and molecular traits 242
by one subgenome over the others, which prompted us to determine the functional 243
divergence of TaSPL8 from 2A, 2B and 2D subgenomes (Wang et al., 2016). We firstly 244
compared the sequence similarity of open reading frames (ORFs) and protein 245
sequences of the three TaSPL8 homoeologous genes, and they showed 97.2% and 246
97.5% similarities for nucleotide and amino acid sequences, respectively. Primer 247
combinations located in the CDS region specific for each homoeolog were designed 248
and verified using the Chinese Spring null-tetrasomic lines of chromosome group 2. 249
We found that the PCR fragments could not be amplified when the corresponding 250
chromosome was deleted (Figure 4A). Using these primers, the transcript level of 251 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
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TaSPL8-2D was observed to be higher than that of TaSPL8-2A and TaSPL8-2B in the 252
lamina joint (Figure 4B). 253
Possible divergence among these homoeologs in their contribution to the observed 254
phenotype was investigated by generating single, double, and triple mutants for each 255
homoeolog. As mentioned above, we firstly obtained the triple mutant 256
TaSPL8-2a/2b/2d by gene editing. The KO-2-1, KO-3-1, and KO-7-1 mutants with 257
three TaSPL8 homoeologs knocked out simultaneously showed erect leaves at both 258
vegetative and reproductive stages (Figure 4C and 4D), with a notable decrease in flag 259
leaf angle compared to the wild-type LC10 (Figure 4D). Additionally, the lamina joint 260
was severely affected and the boundary between the blade and sheath was absent in 261
these KO lines (Figure 4E and 4F). Similar phenotypes were also observed for the KO 262
lines in CB037 and Bobwhite recipients (Supplemental Figure S3). The triple mutant 263
KO-2-1 was crossed to wild-type LC10 to generate single and double mutants. 264
Compared to LC10, the TaSPL8-2a/2B/2D single mutant did not show differences in 265
lamina joint length, while the TaSPL8-2A/2b/2D and TaSPL8-2A/2B/2d single mutants 266
had a shorter lamina joint, with the major effect detected in TaSPL8-2A/2B/2d (Figure 267
4G and 4H). Accordingly, the double mutant TaSPL8-2a/2B/2d had a smaller lamina 268
joint length compared with the TaSPL8-2a/2b/2D double mutant, indicating that the 269
TaSPL8-2D homeolog provides the major contribution for the lamina joint length 270
phenotype (Figure 4G and 4H). Observations from double mutants also corroborated 271
the information from single mutants that TaSPL8-2B affects lamina joint length to a 272
greater extent than TaSPL8-2A (Figure 4G and 4H). These results indicated that the 273
homoeologous gene from the D subgenome contributed to lamina joint length to a 274
greater extent than those from the A and B subgenomes. 275
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To evaluate the potential of manipulating TaSPL8 for optimizing wheat leaf 277
architecture and improving grain yield, we compared the yield-related traits of the 278
TaSPL8 mutant KO-2-1 and the wild-type LC10 in different planting populations of 279 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
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300, 450, 600, and 750 seeds/m2 in two different locations (Beijing and Yangling). 280
Spike numbers per plant for the KO-2-1 line were higher compared to LC10, especially 281
at dense planting populations (450, 600, and 750 seeds/m2) (Figure 4I), which could 282
contribute to higher grain yield. Other traits, including plant height, spike length, grain 283
number per spike, grain weight, heading date (flowering time), and the grain-filling 284
period (time to maturity) did not differ between the KO-2-1 line and LC10 285
(Supplemental Table S1). 286
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Natural variation of TaSPL8-2D 288
To determine the variation of TaSPL8-2D in a natural wheat population, 192 wheat 289
cultivars were screened for nucleotide polymorphisms. No single nucleotide 290
polymorphism was identified in the coding and promoter region, suggesting that the 291
TaSPL8-2D sequence is conserved because of its pivotal role in controlling lamina joint 292
development. The TaSPL8 expression level and lamina joint length was also assessed 293
in 78 wheat cultivars, including 29 cultivars with larger lamina joints (1.5-2.6 cm) and 294
49 cultivars with smaller lamina joints (0.8-1.5 cm) (Supplemental Table S2). The 295
results indicated that the TaSPL8 mRNA level in the lamina joint is higher in cultivars 296
with larger lamina joints compared to those with smaller lamina joints (Figure 4J). 297
These results further corroborated the role of TaSPL8 in lamina joint development. 298
299
TaSPL8 regulates lamina joint development through the auxin and 300
brassinosteroid pathways 301
To understand the mechanisms behind TaSPL8-mediated lamina joint regulation, we 302
performed a transcriptome analysis using RNA-seq in the lamina joint of main tiller 303
from TaSPL8-2a/2b/2d (two independent KO lines: KO-2-1 and KO-7-1) and 304
TaSPL8-2A/2B/2d (single KO line) mutants, as well as of LC10 wild-type plants at the 305
heading stage. For each sample, three biological replicates were employed. The 306
TaSPL8-2A/2B/2d mutant was included in the experiments because the triple 307 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
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TaSPL8-2a/2b/2d mutant lacks the lamina joint and the cell type between the sheath 308
and blade are completely different with respect to LC10, while the single mutant 309
exhibits a phenotype more similar to the wild type, but with a noticeable decrease in 310
lamina joint length. The results indicated that 5980 genes are down-regulated and 2986 311
genes are up-regulated in the TaSPL8-2a/2b/2d mutants compared to LC10 (Figure 5A) 312
(Supplemental Table S3, Supplemental Table S4). Furthermore, a comparison between 313
the TaSPL8-2A/2B/2d single mutant and the wild type indicated that 1841 genes are 314
down-regulated and 1509 are up-regulated (p-values<0.05, |Fold Change|>2). Among 315
the differentially expressed genes (DEGs), 408 and 1310 genes are up-regulated and 316
down-regulated, respectively, in both the triple and single TaSPL8-2D mutant (Figure 317
5A). 318
319
Gene ontology (GO) functional annotation of the DEGs shared between the triple and 320
single TaSPL8 mutants revealed statistically significant enrichment in down-regulated 321
genes for pathways related to ‘response to auxin’, ‘metabolic process for carbohydrate’, 322
‘cellular glucan’, ‘xyloglucan’, ‘biosynthetic process for fatty acid’, ‘cellulose’ and 323
‘cell wall’ (Figure 5B). Among the up-regulated genes, we found an enrichment in 324
pathways associated with protein phosphorylation and photosynthesis (Figure 5B). For 325
cell component ontology, down-regulated genes were enriched in the extracellular 326
region and apoplast and cell wall, while up-regulated genes were enriched in 327
photosystems I and II (Figure 5C). These observations suggest TaSPL8 involvement in 328
cell wall development through the auxin pathway. 329
330
Among the DEGs in the single and triple TaSPL8 mutants, we found 188 331
down-regulated genes related to auxin signaling, including the auxin-responsive 332
Aux/IAA gene family (IAAs), auxin responsive factors (ARFs), auxin efflux carrier 333
components (PINs), small auxin-upregulated RNA (SAUR), and GH3 family 334
(Supplemental Table S3, Supplemental Table S4, Supplemental Figure S4). 335 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
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Measurement of the IAA content in the lamina joint region (tissue between the blade 336
and sheath) of LC10 and the TaSPL8-2a/2b/2d mutant indicated that the IAA content 337
was similar between the mutant and the wild type (Figure 5D), indicating that TaSPL8 338
does not affect IAA biogenesis but may act through auxin response signaling. 339
340
Another interesting category of DEGs that were down-regulated in the single and triple 341
TaSPL8 mutants include genes involved in cell division, cell elongation, and the 342
cytoskeleton (Supplemental Table S3, Supplemental Table S4, Supplemental Figure 343
S4). Downregulation of these genes may lead to failure of cell elongation and result in 344
the lamina joint defects observed in the mutants. These effect can be envisaged 345
especially for the 128 genes encoding glycosyl hydrolase (which function in primary 346
cell wall structure (Lópezbucio et al., 2002)), the 48 genes for expansins (which 347
regulate cell wall extension (Cosgrove, 2015)) and 7 genes for pectinacetylesterase 348
(which function in cell wall extensibility by enhancing aggregation processes between 349
galacturonan chains inside the apoplasm (Sénéchal et al., 2014)). 350
351
Finally, among the down-regulated DEGs, we found three genes with high similarity to 352
rice BRD1/OsDWARF/OsBR6ox, BRD2, and Cytochrome P450 90D2 (D2/CYP90D2), 353
which are implicated in BR biosynthesis processes (Supplemental Table S3, 354
Supplemental Table S4, Supplemental Figure S4). Moreover, 14 genes encoding 355
BRASSINOSTEROID INSENSITIVE 1 (BRI1)-associated proteins and 356
BRASSINAZOLE-RESISTANT 1 (BZR1) homolog proteins, which are responsive to the 357
BR signaling pathway, were also among the down-regulated DEGs (Supplemental 358
Table S3, Supplemental Table S4, Supplemental Figure S4). In agreement with these 359
observations, we found that the brassinolide and castasterone content in the 360
TaSPL8-2a/2b/2d mutant is reduced in comparison with LC10 (Figure 5E), indicating 361
that TaSPL8 affects BR biosynthesis and signaling pathways. All together, our findings 362
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14
indicate that TaSPL8 might regulate lamina joint development through BR and auxin 363
pathways, which are essential for both cell elongation and cell proliferation. 364
365
TaSPL8 acts as a transcriptional activator of TaARF6 and TaD2 366
Since maize LG1 is expressed in ligule and activates PIN1a expression as a 367
transcription activator (Johnston et al., 2014), we screened the down-regulated DEGs 368
identified in our transcriptome analysis for the presence of the 16-nt sequence 369
containing a GTAC core motif (Motif ID: MA0578.1) in their putative promoters, 370
which was previously identified as the cis-element of AtSPL8 targets in Arabidopsis 371
(Kropat et al., 2005). Among the 5980 down-regulated genes in the TaSPL8-2a/2b/2d 372
mutant, 1687 have this motif in their putative promoter sequence, as tested using FIMO 373
(http://meme-suite.org/tools/fimo). The ability of TaSPL8 to bind to the sequence 374
containing the GTAC core motif was assessed using electrophoretic mobility shift 375
assays (EMSAs). To this end, nine probes were synthesized from the promoters of three 376
auxin-responsive genes and four cell expansin genes. The results indicated that TaSPL8 377
can bind to these probes (Supplemental Figure S5). 378
379
Among the DEGs down-regulated in the TaSPL8 mutant, one gene 380
TraesCS5B01G039800 (TaARF6) is the putative ortholog of Arabidopsis thaliana 381
AtARF6 (Supplemental Figure S6). ARFs are key players in mediating auxin-induced 382
gene activation (Lau et al., 2008). In addition, we found that TraesCS5B01G039800 is 383
up-regulated in wild-type LC10 plants after exogenous auxin treatment, while it does 384
not respond to auxin in the KO-2-1 (TaSPL8-2a/2b/2d) mutant (Figure 6A). Therefore, 385
we investigated whether TaSPL8 acts in auxin signaling by directly activating the 386
expression of ARF genes. Similarly, we also tested whether TaSPL8 activates the 387
expression of genes involved in BR biosynthesis because TraesCS3A01G103800 388
(TaD2), the putative ortholog of rice D2, is strongly down-regulated in TaSPL8 389
mutants. Rice D2 encodes a cytochrome P450, which catalyzes the steps from 390 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
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15
6-deoxoteasterone to 3-dehydro-6-deoxoteasterone and from teasterone to 391
3-dehydroteasterone in the late BR biosynthesis pathway. The results of our EMSAs 392
revealed that binding of a TaSPL8 recombinant protein to the putative promoters of 393
TaARF6 and TaD2 occurs in vitro (Figure 6B and 6C). 394
395
In order to determine if the direct binding also takes place in vivo, we first generated a 396
transgenic wheat line (ProUbi::TaSPL8-9MYC) overexpressing a TaSPL8 chimeric 397
protein fused to a Myc tag and then performed chromatin immunoprecipitation (ChIP) 398
assays with a Myc-specific antibody. We achieved three independent transgenic lines 399
(OE-5, OE-10, and OE-12) with high levels of TaSPL8 mRNA and TaSPL8-9Myc 400
protein (Figure 6D). Compared to the wild type of reference (CB037), the OE lines 401
were smaller and displayed fewer tiller numbers at the seedling stage, and decreased 402
spike length and grain size (Supplemental Figure S7). ChIP followed by quantitative 403
PCR (qPCR) was performed from the lamina joint tissue of the OE lines and wild-type 404
CB037. The results indicated that TaSPL8-MYC enrichment of the GTAC core motif 405
of TaARF6 and TaD2 in OE lines is significantly higher than that in wild-type CB037 406
and its enrichment in another region (outside GTAC core motif) is very low, suggesting 407
that TaSPL8 specifically binds to the promoter region of TaARF6 and TaD2 with the 408
GTAC core motif (Figure 6E). The effect of TaSPL8 binding to the TaARF6 and TaD2 409
promoter was investigated using a transient expression assay. Specifically, a plasmid 410
containing the promoter of TaARF6 (pTaARF6::LUC) or the TaD2 promoter 411
(pTaD2::LUC) driving the expression of the luciferase (LUC) reporter gene was 412
co-transformed in tobacco (Nicotiana tabacum) leaves with the effector plasmid 413
(35S::TaSPL8) expressing TaSPL8. The results indicated that TaSPL8 can activate the 414
expression of the reporter for both promoters (Figure 6F). Overall, these findings 415
indicate that TaSPL8 directly binds to the promoter of TaARF6 and TaD2 in the region 416
containing the GTAC motif, which is also associated with their transcriptional 417
activation. Therefore, we can envisage that one of the mechanisms underlying 418 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
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16
TaSPL8-mediated regulation of lamina joint development involves the direct activation 419
of key genes of auxin signaling and BR biogenesis. 420
Discussion 421
TaSPL8 and its homolog LG1 in maize and rice could be a robust target for plant 422
architecture improvement in grass 423
Erect leaves improve light interception and biomass production under field conditions, 424
resulting in increased grain yield in grass crops (Sakamoto et al., 2006). The erect-leaf 425
phenotype of a rice BR-deficient mutant, osdwarf4-1, is associated with enhanced grain 426
yields under dense planting populations, even without extra fertilizer (Sakamoto et al., 427
2006). Therefore, it can be assumed that a compact plant architecture mediated by erect 428
leaves is also an important goal for wheat breeding, but no potential target genes for 429
breeding programs have been identified to date in wheat. This study provides 430
information about the role and mechanisms of TaSPL8, which encodes an SPL-like 431
transcription factor, in controlling leaf angle by regulating lamina joint development. 432
Lamina joint formation is impaired in TaSPL8 knock-out mutants, resulting in erect 433
leaves and a more compact architecture. Our findings suggest that TaSPL8 is an 434
important regulator of compact architecture in wheat, and therefore, could be used as a 435
target to achieve elite wheat cultivars with erect leaves through genetic engineering and 436
molecular breeding. 437
438
TaSPL8 plays a similar function in lamina joint development as LG1 in maize and rice. 439
The mutation of LG1 in maize and rice also resulted in an erect leaf phenotype with a 440
complete loss of the auricle and ligule (Moreno et al., 1997; Lee et al., 2007). Although 441
the contribution of maize and rice LG1 to potential yield in high density planting is still 442
unknown, we speculated that this gene might be a robust target for compact plant 443
architecture in grass, considering the highly conserved function in lamina joint 444
development. However, we have to consider the functional variation among LG1 genes 445 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
17
of maize, rice, and wheat. Beside affecting ligule development, OsLG1 regulates 446
panicle closure or spreading by controlling cell proliferation at the panicle pulvinus 447
(Ishii et al., 2013; Zhu et al., 2013). ZmLG1 also controls the branch angle of the tassel 448
and its knockout mutants result in a more compact tassel than their wild-type 449
counterparts (Bai et al., 2012). Different from OsLG1 and ZmLG1, TaSPL8 does not 450
affect the spike structure, while directly or indirectly leading to the alteration of spike 451
numbers. TaSPL8 mutants show increased spike numbers especially under dense 452
planting condition. This phenotype may be due to erect leaves that allow greater 453
penetration of light to the lower leaves, thereby improving canopy photosynthesis, 454
which in turn, improves the formation of tillers or the ability to generate a spike. A 455
similar phenotype was observed in the osdwarf4-1 rice mutant with erect leaves due to 456
BR deficiency (Sakamoto et al., 2006). Alternatively, TaSPL8 may directly regulate 457
tiller and spike formation. TaSPL8 is highly expressed both in the lamina joint and 458
young spikes and overexpressing plants show a clear reduction in tiller number and 459
spike size. However, an overexpression transgenic line driven by the Ubi promoter 460
actually shows an ectopic effect from the early growth stage. Ubiquitous 461
overexpression may affect the natural function of a gene/protein, for example by 462
altering transcription factor binding specificity when binding is sensitive to doses, or by 463
altering the stoichiometric composition of a multiprotein complex, thus affecting its 464
possible activity and specificity. Therefore, ubiquitous overexpression mutants are not 465
the best for judging a gene’s function, because pleiotropic and “artificial” effects are 466
more likely to be observed. The specific upregulation of TaSPL8 in spikes or tillers will 467
help us to understand its biological function. 468
469
A wild species can be changed to a new form to meet human needs through crop 470
domestication. OsLG1 has experienced substantial artificial selection during rice 471
domestication (Ishii et al., 2013; Zhu et al., 2013). The selection of an SNP residing in 472
the cis-regulatory region of the OsLG1 gene was responsible for the transition from a 473 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
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18
spread panicle typical of ancestral wild rice to the compact panicle of present cultivars 474
during rice domestication (Zhu et al., 2013). This selection led to the reduced 475
expression of OsLG1 specifically at the panicle pulvinus while maintaining normal 476
levels during ligule formation (Zhu et al., 2013). In wheat, we found that the level of 477
TaSPL8 mRNA in the lamina joint is higher in modern cultivars with larger lamina 478
joints (n=49) compared to those with smaller lamina joints (n=29) (Figure 4J), 479
indicating that TaSPL8 is also selected for leaf angle during wheat breeding. However, 480
we did not find sequence variation in the cis-regulatory region of TaSPL8 among these 481
cultivars. Hence, genetic variation in possible distal regulatory elements or epigenetic 482
variation in the TaSPL8 promoter may account for the observed difference in the 483
transcript level. 484
485
TaSPL8-2D is a major contributor to erect leaf in wheat 486
Common wheat is an allohexaploid (BBAADD) derived from a hybridization between 487
a cultivated form of allotetraploid wheat Triticum turgidum (BBAA) and diploid goat 488
grass Aegilops tauschii (DD). Triticum turgidum originates from the hybridization and 489
polyploidization of the diploid species Triticum urartu (AA) and Aegilops speltoides 490
(SS genomes, most probably the donor of the B genome). The combination and 491
interaction of subgenomes in the allohexaploid give rise to phenotypic variation during 492
wheat evolutionary history. The evolution of duplicated gene loci in allopolyploid 493
plants contribute either to a favorable effect of extra gene dosage or to build-up 494
asymmetric expression divergence: only one genome is active, while the homoeoalleles 495
on the other genome(s) are either eliminated or partially or completely suppressed by 496
genetic or epigenetic means (Feldman et al., 2012). However, there is no evidence 497
showing how asymmetric expression divergence affects the trait. We found that the 498
expression level of homoeoallele TaSPL8-2D was higher in the lamina joint compared 499
to the expression level of TaSPL8-2A and TaSPL8-2B. Observations from single, 500
double, and triple mutants also corroborated the information that the TaSPL8-2D 501 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
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19
homeolog provides the major contribution for the lamina joint length phenotype and 502
TaSPL8-2B affects lamina joint length to a greater extent than TaSPL8-2A. These data 503
expand our knowledge about genome asymmetry during allopolyploid wheat evolution 504
and suggest a possible mechanism and evolutionary advantage of this divergence, but 505
addressing this requires further work. 506
507
The utilization of TaSPL8 for compact plant architecture in wheat breeding 508
Simultaneous mutations of all three TaSPL8 homeologs leads to the absence of the 509
lamina joint, sometimes resulting in flag leaves wrapping around the spikes, which 510
blocks spike extension. Thus, potential routes for TaSPL8 utilization in wheat breeding 511
include: 1) using a single mutation of TaCAP-2D or TaCAP-2B to obtain a shorter 512
lamina joint without affecting spike extension, and 2) using alleles associated with 513
lower expression of TaSPL8 and shorter lamina joints, which could be used in 514
marker-assisted selection to fine-tune lamina joint length and leaf angle. 515
516
TaSPL8 modulates leaf angle through auxin and brassinosteroid signaling 517
LG1 orthologs in maize, rice, and wheat function as key regulators of lamina joint 518
development. ZmLG1 was found to function downstream of maize WAB, a TCP 519
transcription factor (Lewis et al., 2014) and to regulate a series of downstream genes 520
including PIN1a, BEL14, and BEL12, which were identified by comparing transcript 521
accumulation between an lg1 mutant and wild type in the preligule region (Johnston et 522
al., 2014). Our study provides evidence that TaSPL8 affects lamina joint development 523
by directly activating the expression of genes involved in auxin and BR pathways. 524
Previous findings indicated that these hormones and various transcription factors 525
contribute to cell division and elongation at the lamina joint region (Sakamoto et al., 526
2006; Bian et al., 2012; Zhao et al., 2013). Here, we have identified the mechanistic link 527
between one transcription factor and the auxin and BR pathways, which are involved in 528
lamina joint development and leaf architecture. Specifically, we found that TaSPL8 529 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
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20
directly binds and activates TaARF6, which is involved in the auxin signaling pathway, 530
and TaD2, which regulates BR biosynthesis (Figure 7). Auxin is essential for cell 531
elongation in nearly all developmental processes. The two Arabidopsis TaARF6 532
orthologs, ARF6 and ARF8, are required for cell elongation in the inflorescence stem, 533
stamen filament, stigmatic papillae, and hypocotyls, and in anther dehiscence (Nagpal 534
et al., 2005; Oh et al., 2014; Liu et al., 2018a). TaARF6 may similarly act as a regulator 535
of cell elongation in the lamina joint. This scenario is supported by findings that other 536
members of the ARF gene family regulate leaf angle. OsARF19-overexpressing rice 537
lines show multiple phenotypes, including increased leaf angles (Zhang et al., 2015), 538
and an OsARF11 loss-of-function line exhibits a reduced angle of the flag leaves 539
(Sakamoto et al., 2013). Similarly, rice mutants with impaired expression of d2, the 540
ortholog of wheat TaD2, are deficient in BR biogenesis and have erect leaves (Hong et 541
al., 2003). This strongly supports our findings that another form of TaSPL8-mediated 542
regulation of lamina joint development occurs through transcriptional activation of 543
TaD2 and the subsequent increase in BR biosynthesis. Although rice BR-deficient 544
mutants are usually dwarfed with short internodes, TaSPL8 mutants do not exhibit 545
other phenotypic alterations except for lamina joint development. We speculate that 546
high expression of TaSPL8 in lamina joint tissue specifically regulates BR biogenesis 547
in this region. Our results indicate that TaSPL8 also affects the expression of genes 548
involved in cell elongation and expansion. For example, we found that 48 genes for 549
expansins are down-regulated in the TaSPL8-2a/2b/2d mutant, and TaSPL8 can bind to 550
the GTAC motif of the promoters of four cell expansin genes in vitro (Supplemental 551
Figure S5). This suggests that TaSPL8 regulates lamina joint development by directly 552
targeting expansin genes. An alternative and not mutually exclusive possibility is that 553
TaSPL8 regulates expansin genes through auxin signaling, since cell elongation genes 554
are also responsive to auxin (Pacheco-Villalobos et al., 2016). 555
556
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21
In conclusion, this study reveals the mechanisms that link one member of the SPL 557
transcription factor family with hormones in regulating lamina joint development, in 558
that TaSPL8 acts upstream of genes involved in the regulation of lamina joint cell 559
elongation, such as TaARF6 and TaD2, which mediate the auxin signaling pathway and 560
BR biosynthesis, respectively. In addition, our results identify additional genes that 561
may function in lamina joint development (e.g., expansin). These findings will be 562
useful for programs aimed at manipulating wheat architecture for improved grain yield. 563
Methods 564
Plant materials 565
Names and geographical origins of all wheat (Triticum aestivum) cultivars used in this 566
study are listed in Supplemental Table S2. Plant materials used for visible phenotypic, 567
microscopic, and molecular analyses were grown in a greenhouse at a relative humidity 568
of 75% and 26/20°C day/night temperatures, with a light intensity of 3000 lux (Master 569
GreenPower CG T 400W E40, PHILIPS). Wheat cultivars used for seed reproduction 570
and phenotypic analysis were grown in the experimental field of China Agricultural 571
University in Beijing (39°57ʹN, 116°17ʹE), Yangling in the Shannxi province (34°16'N, 572
108°04'E). Lamina joints for gene expression analysis were harvested at the heading 573
stage. For each sample, the lamina joint from three different planting pots was 574
harvested, representing three biological replicates. All samples were immediately 575
frozen in liquid nitrogen and stored at -80°C. The natural wheat population consisted of 576
192 wheat cultivars including Chinese landraces (111), Chinese modern cultivars (42), 577
and foreign cultivars from other countries in Europe, Asia, America, and Australia (39). 578
579
Phenotypic analysis 580
The length of the lamina joint and auricle were measured as the widest section between 581
the leaf blade and the sheath with a Vernier caliper. Flag leaf angle was measured using 582
a protractor between the stem under the spike and the flag leaf midrib. Plant height and 583 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
22
the length of the main spike were measured before harvesting, and ten main spikes were 584
bagged at the same time to measure fertile grain number per spike and grain weight per 585
1000 grains. Spike number per plant was calculated as the average value of ten plants. 586
A randomized complete block design with three replicates was employed for 587
phenotyping in open field experiments. 588
589
Mapping of TaSPL8 590
Fine mapping was performed with an F2 and F2-derived F3 population from a cross 591
between cpa and LC10. Primers used for fine mapping were designed based on the 592
sequence of Chinese Spring (IWGSCv1, http://www.wheatgenome.org/) and Aegilops 593
tauschii (Luo et al., 2017) and listed in Supplemental Table S5. Near-isogenic lines 594
(NILs) in the cpa genetic background containing the LC10 allele (NILLC10) and NILs in 595
the LC10 genetic background containing the cpa allele (NILcpa) were generated through 596
backcrossing LC10×cpa F1 plants to LC10 and the cpa mutant for three generations 597
(BC3), respectively. The introgression fragment between markers 2D-173 and LMC-3 598
was screened at each generation. The BC3F1 plant was self-crossed to generate NILLC10 599
and NILcpa, respectively. 600
601
Plasmid construction and transformation 602
For genome editing via CRISPR/Cas9, a small guide RNA (sgRNA) was designed in 603
the first exon sequence of TaSPL8 using the E-CRISP Design website 604
(http://www.e-crisp.org/E-CRISP/designcrispr.html). Two reverse complementary 605
sgRNA sequences with the BsaI cohesive ends were synthesized. Double-stranded 606
oligonucleotides were annealed by cooling from 100°C to room temperature, followed 607
by insertion into the expression cassette of the pBUE411 vector (Xing et al., 2014). 608
Plasmids were transformed into three wheat cultivars (CB037, LC10, and Bobwhite) 609
using Agrobacterium-mediated (strain EHA105) transformation (Ishida et al., 2015). 610
For TaSPL8 overexpression, the ORF of TaSPL8-2D from CB037 and the 9 Myc 611 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
23
domain sequence were amplified and linked by PCR, then the TaSPL8-9Myc fragment 612
was inserted into the pMWB122 vector (Wang et al., 2017) using SmaI and SpeI 613
cloning sites to achieve the ProUbi::TaSPL8-9Myc construct. Plasmids were 614
transformed into the wheat cultivar CB037 using Agrobacterium-mediated (strain 615
EHA105) transformation (Ishida et al., 2015). 616
617
RT-qPCR analysis 618
Total RNA was extracted from different tissues using TRIzol reagent (Takara) 619
according to the manufacturer's instructions and cDNA was synthesized by a reverse 620
transcription kit (Vazyme Biotech, China). Wheat TaACTIN (TraesCS5B01G124100) 621
was used as an internal control. For reverse transcription quantitative PCR (RT-qPCR), 622
the reaction mixture was composed of the cDNA first-strand template, primers, and 623
SYBR Green Mix (Takara) to a final volume of 10 μl. Reactions were performed using 624
the CFX96 real-time system (Bio-Rad). For technical replicates, the RT-qPCR for each 625
sample was replicated three times. Three biological replicates were performed (one 626
replication is shown). The average values of 2-ΔCT were used to determine the 627
differences in gene expression (Applied Biosystems Research Bulletin No. 2 P/N 628
4303859). 629
630
ChIP-qPCR assay 631
Wheat leaves were cross-linked in fixative buffer (1% volume/volume(v/v) 632
formaldehyde) by vacuum infiltration. Cross-linking reactions were stopped by adding 633
glycine at a final concentration of 0.17 M. Chromatin extracts were sonicated and 634
pre-cleared with Dynabeads protein A agarose (Merck Milipore, Germany) for 1 h. For 635
immunoprecipitation, anti-Myc antibody (Abcam, ab9132) was added and samples 636
were incubated at 4°C overnight. The chromatin-antibody complex was recovered with 637
Dynabeads protein A+G and beads were washed sequentially with low salt wash buffer, 638
high salt wash buffer, LiCl wash buffer, and TE buffer for 5 min each (Yang et al., 639 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
24
2016). The protein/DNA complex was eluted with elution buffer (1% weight/volume 640
(w/v) SDS and 0.1 M NaHCO3). Cross-linking was reversed by adding NaCl at a final 641
concentration of 200 mM, followed by incubation for 5 h at 65°C. Proteins in the 642
resulting complex were removed by proteinase K at 45°C for 1 h. DNA was 643
precipitated in the presence of three volumes of ethanol and one-tenth volume of 3 M 644
sodium acetate at pH 5.2. Precipitated DNA was dissolved in 30 μL 10 mM Tris-HCl, 645
pH 7.5, and treated with RNase (DNase-free). Two independent ChIP assays were 646
performed for each sample. Quantitative PCR (qPCR) was carried out using the CFX96 647
real-time system (Bio-Rad). ChIP values were normalized to their respective DNA 648
input values, and the fold changes in concentration were calculated based on the 649
relative enrichment in the overexpression lines compared with wild-type 650
immunoprecipitates. Primer sequences used in the qPCR analysis are listed in 651
Supplemental Table S5. 652
653
Transient expression in tobacco leaves 654
Dual luciferase reporter assays were performed as previously described (Guan and Zhu, 655
2014). A fragment corresponding to 1800 bp upstream of the transcription start site of 656
TaARF6 was PCR amplified from LC10 genomic DNA and cloned into the pGreenII 657
0800-LUC vector (Hellens et al., 2005), generating the reporter pGreenII 658
pTaARF::LUC and pTaD2::LUC plasmids. The ORF of TaSPL8 was cloned into the 659
pUC18-35S vector generating the effector 35S::TaSPL8 plasmid. Young leaves of 660
4-week-old tobacco (Nicotiana tabacum) plants were co-infiltrated with 661
Agrobacterium (strain GV3101) harboring the desired plasmids in the following 662
combination: pTaARF6::LUC + pUC18-35S (empty vector), pTaARF6::LUC + 663
35S::TaSPL8, pTaD2::LUC + pUC18-35S (empty vector), and pTaD2::LUC + 664
35S::TaSPL8. The activities of firefly luciferase under the control of the TaARF6 665
(pTaARF6::LUC) or TaD2 (pTaD2::LUC) promoter and Renilla luciferase under the 666
control of the 35S promoter (35S::REN) were quantified using the Dual-Luciferase 667 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
25
Reporter Assay system (Promega) with the Synergy 2 Multi-Detection Microplate 668
Reader (BioTek Instruments). Normalized data are presented as the ratio of 669
luminescent signal intensity for the pTaARF6::LUC or pTaD2::LUC reporter and 670
luminescent signal intensity for the internal control reporter 35S::REN from three 671
independent biological samples. 672
673
Western blot 674
Harvested leaves were ground in liquid nitrogen and proteins were extracted with 4× 675
loading buffer (200 mM Tris-HCl, pH 6.8, 40% v/v glycerol, 8% w/v SDS, and 20% 676
v/v β-mercaptoethanol) at 100°C for 5 min. The solution was placed on ice for 5 min 677
and centrifuged for 10 min at 12,000 x g at room temperature. Protein extracts were 678
separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis 679
(SDS-PAGE) and transferred to a PVDF membrane (Bio-Rad 162-0177) for 60 min at 680
300 mA in temperature-controlled conditions. The membrane was blocked using 5% 681
(w/v) defatted milk for 1 h. The membrane and primary antibody (1:1000), dissolved in 682
5% (w/v) defatted milk and 1×TBST (1×TBS with 0.1% v/v Tween 20), were 683
incubated at 4°C overnight. After washing three times for 10 min with 1×TBST, the 684
secondary antibody (1:3000) was added in 5% (w/v) defatted milk-1×TBST solution 685
and incubated for 1 h at room temperature, followed by three washing steps of 10 min 686
with 1×TBST. Two-component reagent Clarity Western ECL Substrate (Bio-Rad 687
170-5060) was used for detection. The signal was detected with x-ray film. 688
689
RNA-seq 690
Total RNA was isolated as reported above. Lamina joint from TaSPL8-2A/2B/2d 691
mutant and LC10, and the corresponding part between the leaf blade and sheath from 692
the TaSPL8-2a/2b/2d mutant at the heading stage were collected. Three replicates of 10 693
plants each were used for each tissue type. Barcoded cDNA libraries were constructed 694
using Illumina Poly-A Purification TruSeq library reagents and sequenced on Illumina 695 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
26
HiSeq 2500 or NovaSeq platforms. About 10 Gb of high-quality 125-bp or 150-bp 696
paired-end reads were generated from each library. The overall sequencing quality of 697
the reads in each sample was evaluated by FastQC (v0.11.5) software 698
(http://www.bioinformatics.babraham.ac.uk). Poor-quality bases were removed using 699
Trimmomatic (v0.36) (Bolger et al., 2014) in its paired end model with parameters: 700
LEADING:3 TRAILING:3 HEADCROP:6 SLIDINGWINDOW:4:15 MINLEN:40. 701
The remaining reads were aligned to the wheat reference genome sequence IWGSCv1 702
(http://www.wheatgenome.org/) using Tohat2 v2.11 with the parameters “--b2-D 20 703
--b2-R 3 -N 3 --read-edit-dist 3”(Kim et al., 2013), and only the uniquely mapped reads 704
were retained for the following analysis. FeatureCounts (v1.5.2) with the parameters 705
“--readExtension5 70 --readExtension3 70 –p –C –s 0” was used to estimate the number 706
of reads mapped to respective high-confidence genes annotated from IWGSCv1 in each 707
sample. Bioconductor package "DESeq2" (Love et al., 2014) was used to perform 708
differential expression analysis between TaSPL8 mutant lines and the wild type, and 709
only the genes with an absolute value of log2 (fold change) ≥ 1 and p-value < 0.05 were 710
considered as DEGs. 711
712
For GO analysis, cDNAs of high-confidence genes were aligned to the protein 713
sequences of Oryza sativa and Arabidopsis using BLASTX with an e-value cut-off of 714
1e-05. Only the best alignment with identity ≥ 50% and aligned length ≥ 30 aa were 715
considered. GO annotations were obtained based on the orthologs with the priority: O. 716
sativa > A. thaliana. GO enrichment analysis of DEGs was implemented using the 717
GOseq R package, which is based on the Wallenius non-central hypergeometric 718
distribution and can adjust for gene length bias (Young et al., 2010). GO terms with 719
FDR < 0.01 were considered as statistically significantly enriched. 720
721
Electrophoretic mobility shift assay (EMSA) 722
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27
The full-length ORF sequence of TaSPL8 was cloned into the BamHI site of the 723
pGEX6P-1 vector to produce the chimeric GST-TaSPL8 protein (primer sequences are 724
listed in Supplemental Table S5). Expression of recombinant protein in Transetta 725
(DE3) E. coli (TransGen, Beijing) was induced with 0.1 mM 726
isopropyl-b-D-thiogalactopyranoside (IPTG) in Luria Bertani (LB) buffer overnight at 727
16°C. Cells were subsequently harvested, washed, and suspended in 30 mL of 728
phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 729
mM KH2PO4) containing 1 mM PMSF and 1/2 a tablet of protease inhibitor cocktail 730
(Roche, Germany). Then, cells were sonicated for 30 min and centrifuged at 13,000 x g 731
for 45 min at 4°C. The supernatant was filtered through a 0.22-μm membrane into a 732
50-mL tube. The supernatant was mixed with 1 mL of GST MAG Agarose Beads 733
(Novagen, USA) and shaken overnight at 4°C. GST beads were washed four times with 734
5 mL PBS and the recombinant proteins were eluted by incubation at 4°C for more than 735
4 hours with 50 mM Tris-HCl (pH 8.0) supplemented with 10 mM reduced glutathione. 736
Protein concentration was determined using a Nano Drop 2000 spectrophotometer 737
(ThermoScientific, USA). 738
The biotin-probe was 5' end labeled with biotin. Double-stranded oligonucleotides used 739
in the assays were annealed by cooling from 100°C to room temperature in annealing 740
buffer (Guo et al., 2018). DNA-binding reactions were performed in 20 μL with 741
1×binding buffer (100 mM Tris, 500 mM KCl, and 10 mM DTT; pH 7.5), 10% (v/v) 742
glycerol, 0.5 mM EDTA, 10 mM ZnCl2, and 50 ng μL-1 poly (dI-dC). Competition 743
analysis was done by adding 5- and 10- fold molar excess of the unlabeled DNA 744
fragment (same sequence used for the labeled probe) to the binding reaction, 5 min 745
prior to the labeled probe. After incubation at room temperature for 30 min, samples 746
were loaded onto a 6% native polyacrylamide gel. Electrophoretic transfer to a nylon 747
membrane and detection of the biotin-labeled DNA was performed with the Light Shift 748
Chemiluminescent EMSA Kit (ThermoScientific, USA) according to the 749
manufacturer’s instructions. 750 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
28
751
Quantification of Endogenous IAA and BR 752
The lamina joint of the wild type and the TaSPL8-2a/2b/2d line were harvested at the 753
heading stage. Quantification of endogenous IAA and BR were performed as 754
previously described (Chen et al., 2012; Ding et al., 2013). Each experiment was 755
performed using three replicates. 756
757
Statistical Analysis 758
The statistical analyses, including Student’s t-test and correlation, were performed by 759
SPSS software version 21 (SPSS Inc./IBM Corp., Chicago, IL, United States). All 760
barcharts and boxplot results were graphically presented using Graph Pad Prism 761
software (version 6.0, San Diego, CA, United States). 762
763
Phylogenetic Analysis 764
For phylogenetic analysis, related protein sequences to TaARF6 and TaD2 in 765
Arabidopsis and rice were selected by employing NCBI BLASTp search using nr (non- 766
redundant protein sequences) database. Their corresponding accession numbers were 767
shown in Supplemental Figure S6. The phylogenetic tree was generated by neighbor 768
joining tree method in MEGA5 software. 769
770
Primers for RT-qPCR and vector construction 771
All primers used in this research are listed in Supplemental Table S5. 772
773
Accession Numbers 774
Accession code: the genomic DNA sequence of TaSPL8 in LC10 has been deposited in 775
GenBank with the accession number MH765574. AUXIN RESPONSE FACTOR 776
TaARF6 gene: TraesCS5B01G039800.1; CYP90D2 TaD2 gene: 777
TraesCS3A01G103800. 778 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
29
779
RNA sequencing data are available at the National Center for Biotechnology 780
Information Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra) under the 781
accession number SRP157960. 782
783
Supplemental Data 784
Supplemental Figure S1. Schematic of genotype of LC10, cpa, NILLC10, and NILcpa. 785
Supplemental Figure S2. A Fragment on Chromosome 2D was Deleted in the cpa 786
Mutant. 787
Supplemental Figure S3. CRISPR/Cas9 Knock-out TaSPL8 Mutants in CB037 and 788
Bobwhite Cultivars. 789
Supplemental Figure S4 Heat Map for the DEGs Involved in Auxin and BR Signaling 790
and Cell Elongation in the Wild Type and TaSPL8 Mutants. 791
Supplemental Figure S5 TaSPL8 in vitro Binding of Sequences Containing the 792
GTAC Motif. 793
Supplemental Figure S6 Phylogenetic Tree of TraesCS5B01G039800.1 (TaARF6) 794
and TraesCS3A01G103800.1 (TaD2) with Arabidopsis or Rice Proteins. 795
Supplemental Figure S7 The Phenotypes of TaSPL8-Myc Overexpression (OE) and 796
Wild-type CB037 Lines. 797
Supplemental Table S1 Morphological Characterization of Wild Type and KO-2-1 798
(TaSPL8-2a/2b/2d) Mutant Plants. 799
Supplemental Table S2. The names and geographical origins of all wheat cultivars 800
used in this research. 801
Supplemental Table S3. Differentially expressed transcripts in the Taspl8-2a/2b/2d 802
mutant and the wild type in lamina joint tissue. 803
804
Supplemental Table S4. Differentially expressed transcripts in the Taspl8-2A/2B/2d 805
mutant and the wild type in lamina joint tissue. 806 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
30
Supplemental Table S5. Primers and Gene IDs for sequences used in this study. 807
808
809
Data availability 810
The authors declare that all data supporting the findings of this study are available 811
within the manuscript and its Supplemental files or are available from the 812
corresponding authors upon request. 813
814
815
Acknowledgments 816
We thank Dr. Qijun Chen (China Agricultural University), Dr. Rentao Song (China 817
Agricultural University), and Dr. Xingguo Ye (Chinese Academy of Agricultural 818
Sciences) for providing the CRISPR-Cas9 vector (pBUE11), the transient expression 819
vectors (pUC18-35S and pGreenII 0800-LUC) and the overexpression vector 820
(pMWB122), respectively. We thank Prof. Mingcheng Luo (University of California, 821
Davis, USA) for providing the Aegilops tauschii genome sequence. We thank Prof. 822
Guihua Bai (Kansas State University, USA) for critical reading of the manuscript. We 823
thank Dr. Yuqi Feng (Wuhan University) for assistance in IAA and BR quantification 824
and Dr. Hongxia Li and Dr. Yu Wang (Northwest A&F University) for help in 825
phenotypic measurements of yield-related traits. We also thank the International Wheat 826
Genome Sequencing Consortium (IWGSC) for providing the wheat reference 827
sequence. This work was supported by the National Key Research and Development 828
Program of China (2016YFD0100801, 2016YFD0101004). 829
830
831
Figure Legends 832
Figure 1. Morphological Comparison of the Wheat Cultivar Liaochun10 (LC10) and 833
the cpa Mutant. 834 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
31
(A) Morphologies of LC10 and cpa plants at the heading stage. Bar = 2 cm. 835
(B) Morphologies of the flag leaf in LC10 and cpa plants at the flowering stage. Bar = 2 836
cm. 837
(C) Flag leaf angle of LC10 and cpa plants at the flowering stage. The double asterisk 838
represents significant differences determined by the two-tailed Student’s t-test at P < 839
0.01. Error bars indicate the Standard Deviation (n=10). 840
(D The lamina joint phenotype of LC10 and cpa plants. Bar = 2 cm. 841
(E) Lamina joint length of LC10 and cpa plants. The double asterisk represents 842
significant differences determined by two-tailed Student’s t-test at P < 0.01. Error bars 843
indicate the Standard Deviation (n=10). 844
(F) Morphology of the adaxial surface by a scanning electron microscope of the lamina 845
joint of the LC10 and cpa flag leaf. Image magnification of the red box in the left part 846
(Bar= 200 μm) is shown on the right (Bar = 60 μm). 847
(G) Quantitative cell number and cell size of lamina joint from LC10 and cpa flag leaf. 848
The cell number from the bottom to the top of the lamina joint was counted manually 849
(n=10) and cells size measured by IMAGEJ (n=15). The double asterisk represents 850
significant differences determined by two-tailed Student’s t-test at P < 0.01. Error bars 851
indicate the Standard Deviation. NS: Not Significant. 852
853
Figure 2. Fine Mapping of the TaCPA Locus and Complementation Test Using 854
Near-Isogenic Lines (NILs). 855
(A) Coarse linkage map of TaCPA on chromosome 2D. Numbers indicate the genetic 856
distance between the adjacent markers. Thick black line indicates partial region of 2D 857
chromosome. 858
(B) High-resolution linkage map of TaCPA. The number of recombinants (labeled as r) 859
between the molecular markers and TaCPA is indicated. Thick black line indicates 860
partial region of 2D chromosome. 861
https://plantphysiol.orgDownloaded on March 22, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
32
(C) Fine mapping of TaCPA. Genotypes and phenotypes of recombinants of five F2 862
plants including (F2-820, F2-1990, F2-4153, F2-5088, and F2-7243) are reported. The 863
name and phenotype of F2 individual plants were labeled in the left and right parts, 864
respectively. Black and white blocks indicate genomic region from LC10 and cpa, 865
respectively. Gray block indicates the heterozygous region. 866
(D-E) Phenotype of the flag leaf angle and lamina joint in cpa and NILLC10 (D) and 867
NILcpa (E) plants. Bar = 1 cm. Single and double asterisks represent statistically 868
significant differences determined by the two-tailed Student’s t-test at P < 0.05 and P < 869
0.01, respectively. Error bars indicate the Standard Deviation (n=10). 870
871
Figure 3. Cloning and Characterization of TaSPL8 and the phenotype of its knockout 872
lines by the CRISPR/Cas9 System. 873
(A) Amino acid sequence alignment of TaSPL8 and its orthologs in rice 874
(Os04g0656500), maize (Zm00001d002005), and sorghum (SORBI_3006G247700). 875
The conserved SBP domain is indicated by a red line. 876
(B) Expression pattern of TaSPL8 in various tissues. The expression levels were 877
normalized to wheat TaACTIN. Each bar in the graph corresponds to the mean value of 878
three data points, which are shown by dots. 879
(C) Sequence of an sgRNA designed to target a conserved region of exon 1 among the 880
three TaSPL8 homoeologs. The protospacer-adjacent motif (PAM) sequence is 881
highlighted in red. 882
(D) The genotypes of three mutant lines were identified by sequencing. "+" and "−" 883
indicate the insertion or deletion caused by CRISPR/Cas9-induced mutations, 884
respectively. Numbers indicate the length of the insertion or deletion. 885
(E) Flag leaf phenotypes of mutant Taspl8-2A/2B/2d, cpa and LC10. Bar=1 cm. 886
(F) Flag leaf angle and lamina joint length of Taspl8-2A/2B/2d, cpa and LC10 plants. 887
The double asterisk represents significant differences determined by two-tailed 888
Student’s t-test at P < 0.01. Error bars indicate the Standard Deviation (n=10). 889 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
33
890
Figure 4. Three homoeologous genes of TaSPL8 differentially contribute to lamina 891
joint growth. 892
(A) The specificity of primers for three TaSPL8 homoeologous genes were verified by 893
amplifying the genomic DNA of the wheat cultivar Chinese Spring null-tetrasomic 894
lines of chromosome group 2, where N2DT2B represents nullisomic2D-tetrasomic2B, 895
for example. 896
(B) Relative abundance of three homoeologous genes, TaSPL8-2A, TaSPL8-2B, and 897
TaSPL8-2D, in the lamina joint. DNA of the Chinese Spring cultivar was used to 898
analyze the amplification efficiency of the three pairs of primers. The RT-qPCR data 899
were normalized to wheat TaACTIN and each bar in the graph corresponds to the mean 900
value of three data points, which are shown by dots. 901
(C-D) Leaf phenotype of LC10 and mutant lines at heading (C) and flowering stages 902
(D). Bar = 2 cm. 903
(E-F) Adaxial (E) and abaxial side (F) phenotypes of the lamina joint region of LC10 904
and mutant lines. Bar = 2 cm. 905
(G) Lamina joint phenotype of TaSPL8 single, double, and triple homoeologous mutant 906
plants. Bar = 1 cm. 907
(H) The lamina joint length of TaSPL8 single, double, and triple homoeologous 908
mutants. The values shown are averages from three plants and the Standard Deviation 909
is indicated. NS: non-significant. Single and double asterisks represent statistically 910
significant differences determined by the two-tailed Student’s t-test at P < 0.05 and P < 911
0.01, respectively. 912
(I) Spike number per plant in LC10 and mutant lines in different planting densities 913
(300, 450, 600, and 750 seeds/m2) in Beijing and Yangling. The values shown are 914
averages from three replicates and the Standard Deviation (SD) is reported. Single and 915
double asterisks represent statistically significant differences determined by the 916
two-tailed Student’s t-test at P < 0.05 and P < 0.01, respectively. 917 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
34
(J) Comparison of TaSPL8 expression levels between the wheat cultivars with larger 918
(1.5-2.6 cm) (n = 29) and smaller (0.8-1.5 cm) (n = 49) lamina joints. The level of 919
TaSPL8 mRNA was normalized to wheat ACTIN. Lines in the box plots indicate the 920
median. The 10th/90th percentiles of outliers are shown. The single asterisk represents 921
statistically significant differences as determined by Student’s t-test at P < 0.05. 922
923
Figure 5. Gene Ontology (GO) Term Enrichment Analysis and IAA/BR Level 924
Measurement in TaSPL8 Mutants and Wild Type Plants. 925
(A) Venn diagram analysis of DEGs in TaSPL8-2A/2B/2d and TaSPL8-2a/2b/2d 926
mutants compared to the wild type (LC10). 927
(B) GO terms for biological processes exhibiting statistically significant enrichment in 928
DEGs (only DEGs in common for both single and triple TaSPL8 mutants were 929
considered). 930
(C) GO terms for cell components exhibiting statistically significant enrichment in 931
DEGs. 932
(D-E) IAA (Indoleacetic acid), BL (Brassinolide), and CS (Castasterone) levels were 933
measured in the lamina joint tissue of LC10 and KO-2-1 (TaSPL8-2a/2b/2d) plants. NS 934
indicates a non-significant difference. Single and double asterisks represent statistically 935
significant differences determined by the two-tailed Student’s t-test at P < 0.05 and P < 936
0.01, respectively. Error bars indicate the Standard Deviation (n=3). NS: Not 937
Significant. 938
939
940
Figure 6. TaSPL8 Binds and Activates TaARF6 and TaD2. 941
(A) TaARF expression analysis after auxin treatment (10-4 mol/L) in the wild-type 942
LC10 plant and a TaSPL8 mutant (KO-2-1). Each bar in the graph corresponds to the 943
mean value of three data points, which are shown by dots. 944
https://plantphysiol.orgDownloaded on March 22, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
35
(B-C) EMSAs to assess the TaSPL8 binding to the promoter region of TaARF6 (B) and 945
TaD2 (C) containing the GTAC motif. "+" and "−" indicate the presence and absence, 946
respectively, of corresponding probes, proteins, or competitors. "1×" and "2×" indicate 947
the amount of proteins in the reaction. "5×" and "10×" indicate 5- and 10-fold molar 948
excess, respectively, of competitor or mutated competitor (MT) probes relative to 949
biotin-labeled probes. 950
(D) Generation of wheat transgenic lines overexpressing TaSPL8-Myc chimeric 951
protein (OE lines) in the CB037 genetic background. The level of TaSPL8 transcripts 952
(left panel) and TaSPL8-Myc protein (right panel) were measured. RT-qPCR data were 953
normalized to wheat TaACTIN and each bar in the graph corresponds to the mean value 954
of three data points, which are shown by dots. Double asterisks represent statistically 955
significant differences determined by the two-tailed Student’s t-test at P < 0.01. 956
(E) ChIP assays to detect TaSPL8 binding of TaARF6 and TaD2 in vivo. Arrows 957
indicate the position (P1 and P2) of primer combinations used in qPCR after ChIP 958
assays. P1 indicates the region where GTAC is located and P2 indicates the region 959
without GTAC with more than 400bp distance from P1. Each bar in the graph 960
corresponds to the mean value of three data points, which are shown by dots. Double 961
asterisks represent statistically significant differences determined by the two-tailed 962
Student’s t-test at P < 0.01. 963
(F) Transactivation assays with tobacco leaves infiltrated with different combinations 964
of effector and reporter constructs. Individual values (black dots) and means (bars) of 965
three independent biological replicates are shown. The double asterisks represent 966
significant differences determined by the two-tailed Student’s t-test at P < 0.01. 967
968
Figure 7. Molecular Model for the Regulation of Wheat Lamina Joint Development by 969
TaSPL8. 970
TaSPL8 directly binds to the TaARF6 and TaD2 promoters to activate their expression. 971
TaARF6 mediates the auxin signaling pathway, while TaD2 is involved in BR 972 https://plantphysiol.orgDownloaded on March 22, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
36
biogenesis. Both hormones are well known for regulating cell elongation, lamina joint 973
development, and leaf angle. 974
975
Supplemental Data 976
Supplemental Figure S1. Schematic of genotype of LC10, cpa, NILLC10, and NILcpa. 977
Black bar, genomic region from LC10; white bar, genomic region from cpa. 978
979
Supplemental Figure S2. A Fragment on Chromosome 2D was Deleted in the cpa 980
Mutant. 981
982
Supplemental Figure S3. CRISPR/Cas9 Knock-out TaSPL8 Mutants in CB037 and 983
Bobwhite Cultivars. 984
Supplemental Figure S4 Heat Map for the DEGs Involved in Auxin and BR 985
Signaling and Cell Elongation in the Wild Type and TaSPL8 Mutants. 986
987
Supplemental Figure S5 TaSPL8 in vitro Binding of Sequences Containing the 988
GTAC Motif. 989
990
Supplemental Figure S6 Phylogenetic Tree of TraesCS5B01G039800.1 (TaARF6) 991
and TraesCS3A01G103800.1 (TaD2) with Arabidopsis or Rice Proteins. 992
993
Supplemental Figure S7 The Phenotypes of TaSPL8-Myc Overexpression (OE) and 994
Wild-type CB037 Lines. 995
Supplemental Table S1 Morphological Characterization of Wild Type and KO-2-1 996
(TaSPL8-2a/2b/2d) Mutant Plants. 997
998
https://plantphysiol.orgDownloaded on March 22, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
37
Supplemental Table S2. The names and geographical origins of all wheat cultivars 999
used in this research. TaSPL8 expression level and lamina joint length in 78 wheat 1000
cultivars are also included. 1001
1002
Supplemental Table S3. Differentially expressed transcripts in the Taspl8-2a/2b/2d 1003
mutant and the wild type in lamina joint tissue. 1004
1005
Supplemental Table S4. Differentially expressed transcripts in the Taspl8-2A/2B/2d 1006
mutant and the wild type in lamina joint tissue. 1007
1008
1009
Supplemental Table S5. Primers and Gene IDs for sequences used in this study. 1010
1011
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