qngr9 and genetic complementation. · supplementary note: map-based cloning of qngr9 and genetic...

Supplementary note:

Map-based cloning of qNGR9 and genetic complementation.

Fine-scale mapping of qNGR9, based on the pattern of segregation among 11,654

BC3F2 progeny bred from the backcross between RIL-D22 (the donor parent) and

QZL2 (the recurrent parent). It was mapped to a ~18.6-kb region flanked by the

molecular markers W13 and W18 (Supplementary Fig. 1a). This region contains

two predicted ORFs (Os09g0441700 and Os09g0441900). Sequence comparison of

this region between RIL-D22 and QZL2 revealed that the former ORF, which

encodes a putative cytochrome P450 protein, had one synonymous polymorphism

(c.996G＞A) and three non-synonymous polymorphisms (c.62A＞G, c.1282G＞C

and c.1526C ＞ T) (Supplementary Fig. 1b). Sequence comparison of

Os09g0441900 ORF between RIL-D22 and QZL2 revealed four polymorphisms:

three non-synonymous SNPs （c.314G＞A, c.683A＞T and c.970A＞T）in the

exon5, and a 625-bp deletion which is matched with the already defined dep1

variant of the DEP1 gene10-12, 35, 36 (Supplementary Fig. 1c).

We developed the near-isogenic lines, NIL-qNGR9, which carries a small NJ6

segment including the qNGR9. NIL-qNGR9 plants exhibited nitrogen-sensitive

vegetative growth (Supplementary Fig. 2a). To confirm that DEP1 and qNGR9 are

synonymous, the dep1-1 cDNA were amplified from QZL2, and then generated

transgenic NIL-qNGR9 plants which were over-expressed the QZL2 dep1-1 cDNA

under the control of the native DEP1 promoter (Supplementary Fig. 2b). We found

that the transgenic NIL-qNGR9 plants over-expressing dep1-1 were semi-dwarfed in

stature as compared to non-transgenic NIL-qNGR9 plants, and the vegetative

growth of the transgenic NIL-qNGR9 plants was largely unresponsive to nitrogen

input level (Supplementary Fig. 2b). Similarly, NIL-dep1-1 plants carrying the

dep1-1 allele from QZL2 exhibited the nitrogen-insensitive vegetative growth

response as compared with NIL-DEP1 plants (Supplementary Fig. 2c). In contrast,

Nature Genetics: doi:10.1038/ng.2958

the transgenic NIL-qNGR9 plants expressing the Os09g0441700 cDNA amplified

from QZL2 did not differ in phenotype from the non-transgenic NIL-qNGR9 plants

(data not shown). Thus, DEP1 and qNGR9 are synonymous.

In the F2 population bred from the cross between NIL-DEP1 and NIL-dep1-1 plants,

individuals which were heterozygous at the DEP1 locus produced semi-dwarf

phenotypes which were intermediate between that of either homozygote, whereas

75 of 288 segregants homozygous for the NJ6 DEP1 allele were tall and largely

responsive to nitrogen input level (data not shown). Thus, the dep1-1 allele from

QZL2 is semidominant.

Identification of the dep1-32 allele

We previously reported that a variant allele of DEP1, dep1, which was found in the

Italian rice cultivar Balilla, conferred the increased number of grains per panicle and

higher grain yield10. To further understand the molecular mechanism underlying

DEP1 and its interaction network, we performed a genetic screening to identify the

dep1-like rice mutants using ethylmethane sulphonate mutagenized population of cv.

Minghui63. We found a dep1-like32 mutant characterized by dense and erect

panicles. We then did the cross between dep1-like32 mutant and Minghui63, and

genetic segregation of the F2 population indicated that dep1-like32 is a recessive

loss-of-function mutation.

Fine-scale mapping of dep1-like32 based on 1,384 F2 segregants bred from the cross

between dep1-like32 mutant and Nipponbare. It was finally mapped to region

flanked by the molecular markers W13 and W17 in Chromosome 9, and this region

contains DEP1 gene (data not shown). Sequence comparison of this region between

dep1-like32 and Nipponbare revealed that DEP1 of the dep1-like32 mutant had one

synonymous polymorphism (c.277G＞T), which caused premature stop (Fig. 2b).

The genetic complementation also confirmed that dep1-like32 was synonymous


with DEP1 (data not shown). Thus, this mutation was renamed as dep1-32.

DEP1 is involved in determining the amount and direction of cell division.

Compared to NIL-DEP1 plants, NIL-dep1-32 plants showed a more pronounced

longitudinal cell elongation in their shortened internode, and produced wider but

also shorter leaves than that formed by NIL-DEP1 plants (Supplementary Fig. 4).

Although the width of the leaf epidermal cells in NIL-dep1-32 plants was

indistinguishable from that of NIL-DEP1 cells, there was a marked reduction in

longitudinal cell proliferation (Supplementary Fig. 4), suggesting that DEP1

participates in determining the amount and direction of cell division, which in turn

controls organ size and shape.

We also found that the longitudinal cell numbers of the shortened internode in

NIL-dep1-1 plants were less than that of NIL-DEP1 plants (Supplementary Fig. 7).

In addition, the transcript abundance of genes (such as CDKA1, CYCD3 and E2F2)

implicated in the determination of cell cycle, was considerably higher in NIL-DEP1

plants than that in the NIL-dep1-1 plants (Supplementary Fig. 8). These results

suggest that the dep1-1 allele functions as a negative regulator of cell proliferation

in control of stem elongation. Thus, the dep1-1 allele appeared to produce two

contrasting effects on plant architecture: repressing longitudinal cell division and

plant height during vegetative growth period, whereas promoting cell proliferation

and panicle branching during the reproductive stage10.

The effect of DEP1 on nitrogen uptake and metabolism.

In rice, ammonium is taken up by high affinity transporters, and most of it is

subsequently assimilated into glutamine by the coupled reaction of glutamine

synthetase (GS) and glutamate synthase (GOGAT) in rice16. The previous studies

have shown that the cytosolic GS1;2 and the plastidic NADH-GOGAT1 are largely

responsible for the primary assimilation of ammonium16. A quantitative real time


PCR based examination of the transcriptional levels of the genes involved in the

uptake and assimilation of nitrogen in the NILs roots were all up-regulated in both

NIL-dep1-1 and NIL-DEP1 plants grown under low nitrogen conditions. However,

the transcript levels of all five key genes (AMT1;1, GS1;1, GS1;2, NADH-GOGAT1

and NADH-GOGAT2) were markedly higher in NIL-dep1-1 than that of NIL-DEP1

roots (Supplementary Fig. 9).

The bulk of the nitrogen accumulated by plants during their period of vegetative

growth (in rice, ~80%) is remobilized and translocated to the developing grain37,

and the major enzymes responsible for this process are cytosolic GS1;1 and

NADH-GOGAT114, 37-38. We found that GS1;1 and NADH-GOGAT1 were clearly

up-regulated in NIL-dep1-1 plants provided with either an ample or a limiting

supply of nitrogen (Supplementary Fig. 10), consistent with the experimentally

demonstrated positive correlation between grain yield and GS1 activity in maize39.

In addition, previous studies indicated the involvement of the plant glutamate-like

receptor homologs (GLRs) in nitrogen metabolism40, we also found that transcript

levels of rice homologs of GLR were up-regulated in the NIL-dep1-1 plants

(Supplementary Fig. 11). These results suggest that DEP1 regulates both plant

developmental responses to nitrogen and nitrogen metabolism, the dep1-1 allele is

involved in nitrogen uptake and assimilation, more than the DEP1 allele from NJ6.

Analysis of transgenic plants over-expressing RGB1.

RGB1 transcript level in four independently transformed 21-day-old seedlings

over-expressing RGB1 (assessed using quantitative real time PCR) was significantly

higher than wild type plants (Supplementary Fig. 18a). Neither tiller number nor

stem elongation in paddy-grown transgenic RGB1 over-expressors responded to

nitrogen input level (Fig. 4d, e and Supplementary Fig. 18b). The flag leaves of

nitrogen-starved NIL-DEP1 plants were pale green, but those of the transgenic

plants overexpressing RGB1 were dark green and their leaf length did not respond


to nitrogen input level (Supplementary Fig. 18c). In addition, nitrogen-starvation

induced root elongation and root/shoot ratio of the latter seedlings remained

unaffected under low nitrogen conditions (Supplementary Fig. 18d). These results

suggest that nitrogen-promoting vegetative growth response is dependent on RGB1

availability or activity.

35, Wang, J. et al. Identification and characterization of the erect-pose panicle gene

EP conferring high grain yield in rice (Oryza sativa L.). Theor. Appl. Genet. 119,

85-91 (2009).

36, Yi, X. et al. Introgression of qPE9-1 allele, conferring the panicle erectness,

leads to the decrease of grain yield per plant in japonica rice (Oryza sativa L.). J

Genet Genomics. 38, 217-223 (2011).

37, Mae, T, & Ohira, K. The remobilization of nitrogen related to leaf growth and

senescence in rice plants (Oryza sativa L.). Plant Cell Physiol. 22, 1067-1074

(1981).

38, Lea, P. J. & Miflin, B. J. Glutamate synthase and the synthesis of glutamate in

plants. Plant Physiol. Bioch. 41, 555-564 (2003).

39, Martin, A. et al. Cytosolic glutamine synthetase isoforms of maize are

specifically involved in the control of grain production. Plant Cell 18,

3252-3274 (2006).

40, Kang, J. & Turano, F. J. The putative glutamate receptor 1.1 (AtGLR1.1)

function as a regulator of carbon and nitrogen metabolism in Arabidopsis

thaliana. Proc. Natl. Acad. Sci. USA 100, 6872-6877 (2003).


Supplementary Tables

Supplementary Table 1. Rice accessions surveyed for variation in NUE

Accession/Variety# Origin Class Changes in

plant height*

Changes in

tillering§

sd1

allele

Guichao2 Guangdong,

China indica 21.2 ± 2.6 7.6 ± 0.5 yes

Aizizhan Guangdong,

China indica 18.2 ± 2.9 8.7 ± 1.6 yes

Yeqingzhan3 Guangdong,

China indica 23.7 ± 3.9 9.2 ± 1.4 yes

Guangluai4 Guangdong,

China indica 17.8 ± 4.6 7.8 ± 0.7 yes

9311 Jiangsu,

China indica 19.8 ± 3.1 9.3 ± 1.5 yes

Minghui63

Fujiang,

China indica 22.7 ± 3.2 11.2 ± 3.4 yes

Zhefu802

Zhejiang,

China indica 15.2 ± 2.9 7.2 ± 0.7 yes

Erjiuqing

Zhejiang,

China indica 27.9 ± 3.7 9.3 ± 1.2 yes

Zhenshan97B

Jiangxi,

China indica 19.1 ± 1.3 7.8 ± 1.4 yes

TN-1

(Taichung Native-1)

Taiwan,

China indica 24.8 ± 4.2 7.2 ± 0.7 yes

IR8 IRRI,

Philippine indica 19.4 ± 2.1 9.8 ± 1.9 yes

IR24 IRRI,


IR36 IRRI,


IR64 IRRI,


IR65600-27 IRRI,


IR66764-60

IRRI,


RD23 Thailand indica 28.6 ± 4.3 8.2 ± 1.7 yes

Bg90-2 Sri Lanka indica 25.7 ± 3.3 7.6 ± 0.9 yes

Amol3 Iran indica 24.8 ± 2.7 9.2 ± 2.1 yes

Khazar Iran indica 19.2 ± 2.3 8.1 ± 1.7 yes

Nantehao Fujiang, indica 17.9 ± 1.9 9.2 ± 2.7 no


China

Lucaihao Fujiang,

China indica 29.8 ± 4.3 11.4 ± 2.1 no

Nanjing6 Jiangsu,

China indica 31.4 ± 3.8 9.8 ± 2.5 no

Dabaigu Guangxi,

China indica 28.9 ± 5.4 10.7 ± 2.3 no

Xiaohongdao Anhui,

China indica 32.2 ± 4.7 12.4 ± 3.9 no

Aus116 Bangladesh indica 35.8 ± 6.6 10.9 ± 1.2 no

Aus143 India indica 37.9 ± 3.5 11.2 ± 1.0 no

Kasalath India indica 34.2 ± 4.5 12.7 ± 1.9 no

30416 Brazil indica 30.2 ± 4.1 11.2 ± 3.1 no

9177 India indica 24.2 ± 3.3 10.2 ± 2.7 no

8231 Vietnam indica 32.7 ± 4.9 11.5 ± 2.4 no

9148 Thailand indica 35.4 ± 6.3 12.4 ± 3.1 no

Laolaiqing Jiangsu,

China japonica 25.8 ± 3.2 7.3 ± 1.6 no

Wuyujing5 Jiangsu,


Wuyunjing7 Jiangsu,


Taihuoqing Zhejiang,


Xiushui04 Zhejiang,


Xiushui110 Zhejiang,


Zhongzao01 Zhejiang,


Zhonghua11 Tianjing,


Xinxiannu1 Henan,


Zhongxin5 Hebei,


Qianzhonglang2 Liaoning,


Weiguo Liaoning,


Liaohe5 Liaoning,


Longjing1 Heilongjiang, japonica 13.5 ± 3.4 4.9 ± 1.6 no


Chgina

Longjing3 Heilongjiang,

Chgina japonica 16.8 ± 2.6 5.2 ± 1.7 no

Jijing62 Jilin,


Jijing88 Jilin,


Yunjing36 Yunnan,


Dianjinyou1 Yunnan,


Baisenugu Guangxi,


Tainong54 Taiwan,


Tainong67 Taiwan,


Taizhong65 Taiwan,


Katy USA japonica 15.2 ± 2.7 7.0 ± 1.2 no

Lemont USA japonica 17.2 ± 1.5 7.2 ± 0.6 no

Nongken58 Japan japonica 15.8 ± 2.3 5.3 ± 1.1 no

Shanxin22 Japan japonica 14.9 ± 1.7 5.1 ± 2.0 no

Nipponbare Japan japonica 17.2 ± 2.6 5.8 ± 0.4 no

19282 Egypt japonica 16.9 ± 3.6 5.6± 1.2 no

Balila Italy japonica 12.9 ± 4.7 3.9 ± 1.6 no

#On the basis of Garris et al. (2005), the major groups of Asian cultivated rice were

selected, including aus (AUS), indica (IND), temperate japonica (TEJ) and

tropical japonica (TRJ)14-15. *Difference in plant height of the rice plants grown under the two contrasting

nitrogen input levels (low nitrogen, 30 kg/ha; high nitrogen, 300 kg/ha). §Difference in tiller numbers of rice plants grown under the two contrasting

nitrogen input levels (low nitrogen, 30 kg/ha; high nitrogen, 300 kg/ha).


Supplementary Table 2. The effect of nitrogen fertilization levels on plant

architecture and harvest index.

Variety Nitrogen

fertilization (kg/ha)Plant height

Tiller number per

plant

Harvest

index

QZL2 0 76.5 ± 0.6 8.2 ± 0.2 0.55 ± 0.03

60 77.8 ± 04 8.7 ± 0.3 0.59 ± 0.02

200 78.2 ± 0.2 8.9 ± 0.4 0.63 ± 0.01

300 80.6 ± 0.4 9.2 ± 0.3 0.62 ± 0.02

NJ6 0 106.3 ± 0.7 7.0 ± 0.3 0.47 ± 0.02

60 111.7 ± 1.2 9.5 ± 0.2 0.46 ± 0.03

200 128.3 ± 0.7 12.3 ± 0.4 0.44 ± 0.01

300 136.4 ± 0.9 14.8 ± 0.6 0.45 ± 0.04

RIL-D04 0 75.8 ± 1.0 5.6 ± 0.1 0.57 ± 0.03

60 78.0 ± 0.7 5.8 ± 0.2 0.58 ± 0.01

200 81.8 ± 0.5 6.0 ± 0.2 0.60 ± 0.02

300 80.9 ± 1.2 6.2 ± 0.2 0.59 ± 0.03

RIL-D22 0 82.8 ± 0.8 3.2 ± 0.4 0.48 ± 0.02

60 86.3 ± 1.1 5.6 ± 0.3 0.49 ± 0.03

200 102.8 ±1.3 8.0 ± 0.3 0.48 ± 0.01

300 118.1± 0.6 10.4 ± 0.5 0.49 ± 0.04

*Rice plants were spaced 20 cm apart from one another in a paddy field managed

in conventional fashion. Data shown as mean ± SE (n = 60).


Supplementary Table 3. Primer sequences used for the fine-scale genetic mapping of qNGR9.

Primer Forward sequence Reverse sequence Restriction

enzymes

RM5112 CTCGCAATTTATACGTAATC CTCACGAAATAAAATGAGTG

RM3700 AAATGCCCCATGCACAAC TTGTCAGATTGTCACCAGGG

RM7424 AGAAGCCCATCTAGCAGCAG TCAAGCTAGCCACACAGCTG

RM7048 CAACCCCTAATTTCACGCTC GACTTCACTGGCACTGGATG

RM257 CAGTTCCGAGCAAGAGTACTC GGATCGGACGTGGCATATG

W3 CACGCTTCCTAAACTACTAAAC CTGACACCGGTCATTACTCTAT

W5 TAGCGAGGATGGCATGTGAAT GGCTAAGGAAAGACGGAAAGTC

W6 GTGCTTCAACTGCCTGCGAGAC TGTGGCTTTGCTCCTATTTGTT

W10-2 AATGAGTTACTGAAAGCATACGG ATGACTGTTGTTGGTTGCATAA

W14-2 TCGGCTAATGCCAGGACT GTTATTTCTTATGTGACGGATG

W18 CAAGAAGCAATTAACTCACTCA AATGAAATATCGAAAGAAGAGG

W21 GATGGACACTTGTTATCTTCTC CATAAGAACTGGAAGTTTGTAA

W17 CTCTGGCATCGGCTACTCAC GGGCTGTTTGGATCAGGGAC HaeIII

W13 CCCTACCGTGGCTTCTTGTCC TCTGCCTCCCATGTCCTAACTCG

W23 AGCTACTTATCAAACAGCCCCACC ATCGCATGGAAAGAATCGGTGA


Supplementary Table 4. Primer sequences used for building DNA constructs.

Primers Forward sequence(5-3) Reverse sequence(5-3)

GFP CCCCCGGGATGGTGAGCAAGGGCGAGGAGCT CGGGATCCCTTGTACAGCTCGTCCATGCCGT

GFP-dep1 CGGGATCCCTCGAGGTCGACTGACCAATG GCTCTAGAGATGTTGAAGCAGGTGCAGCT

AD12-OX GCTCTAGAATGGCGTCCGTGGCGGAGCTCAA GCGTCGACTCAAACTATTTTCCGGTGTCCGCT

AD12-RNAi GCTCTAGAGGGTTCATGTGATGCAACTG GCGTCGACCCCCTCATGAGAGTTTTGGA

AD12-GFP CCCGGGTATGGCGTCCGTGGCGGAGCT GCTCTAGAAACTATTTTCCGGTGTCCGCT

RGA-GFP TCCCCCGGGATGTCCGTGCTTACCTGTG GCTCTAGAAGTTCCTTCCCTGGAG

HA-RGA1 CGGAATTCCTATGTCCGTGCTTACCTGTG GCGGATCCTCAAGTTCCTTCCCTGGAG

RGG1-GFP TCCCCCGGGATGCAGGCCGGAGGAGGAGG TCCCCCGGGTCACAAAAACCAGCATTTGC

HA-RGG1 CGGAATTCCTATGCAGGCCGGAGGAGGAGG GCTCTAGACAAAAACCAGCATTTGC

RGG2-GFP TCCCCCGGGATGAGGGGGGAGGCGAACGG GCTCTAGAGGAAAAATCTGAGCCT

HA-RGG2 GCGAATTCCTATGAGGGGGGAGGCGAACGG GCGGATCCCTAGGAAAAATCTGAGCCT

BiFC-DEP1-1 GCGTCGACAATGGGGGAGGAGGCGGTGGTG GCGGATCCTCAACATAAGCAACCACTGAG

BiFC-YN-DEP1 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGTGAGCAAGGGCGAGG GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAACATAAGCAACCACTGAG

BiFC-YC-DEP1 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGACAAGCAGAAGAAC GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAACATAAGCAACCACTGAG

BiFC-dep1-1 GCGTCGACAATGGGGGAGGAGGCGGTGGTG GCGGATCCCTAGATGTTGAAGCAGGTGCAG

BiFC-YN-dep1 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGTGAGCAAGGGCGAGG GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGATGTTGAAGCAGGTGCAG

BiFC-YC-dep1 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGACAAGCAGAAGAAC GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGATGTTGAAGCAGGTGCAG

BiFC-YN-DEP1-1-56 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGTGAGCAAGGGCGAGG GGGGACCACTTT GTACAA GAAAGCTGGGTCTCAAGAAACGGGCTGAGCTC

BiFC-YC-DEP1 1-56 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGACAAGCAGAAGAAC GGGGACCACTTT GTACAA GAAAGCTGGGTCTCAAGAAACGGGCTGAGCTC

BiFC-YN-DEP1-1-72 GGGG ACA AGT TTGTACAAAAAAGCAGGCTTCATGGTGAGCAAGGGCGAGG GGGGACCACTTT GTACAAGAAAGCTGGGTCTCATCAATGTTTTGTACCAACAAACT

BiFC-YC-DEP 1-72 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGACAAGCAGAAGAAC GGGGACCACTTTGTACAAGAAAGCTGGGTCTCATCAATGTTTTGTACCAACAAACT

BiFC-YN-DEP1-Δ56-63 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGTGAGCAAGGGCGAGG GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAATGTTTTGTACCAACAAACT


GTTGGTACAAAACATACAAAGAGAAGGAGGCACAG GCGGATCCTCAACATAAGCAACCACTGAG

BiFC-YC-DEP1Δ56-63 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGACAAGCAGAAGAAC GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAATGTTTTGTACCAACAAACT


BiFC-YN-DEP1Δ56-72 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGTGAGCAAGGGCGAGG GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAATGTTTTGTACCAACAAACT

GGAGCTCAGCCCGTTGACCCACTAATACCAACAAA GCGGATCCTCAACATAAGCAACCACTGAG

BiFC-YN-DEP1Δ56-79 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGTGAGCAAGGGCGAGG GGGG ACCACTTT GTACAAGAAAGCTGGGTCTCAATGTTTTGTACCAACAAACT

GGAGCTCAGCCCGTTAGAAGGAGGCACAGATCTTG GCGGATCCTCAACATAAGCAACCACTGAG

BiFC-DEP1Δ72-1 CGGTCGACAATGGGGGAGGAGGCGGTGGT GCGGATCCTCAACATAAGCAACCACTGAG

BiFC-YN-DEP1Δ72 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGTGAGCAAGGGCGAGG GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAACATAAGCAACCACTGAG

BiFC-DEP1Δ79-1 CGGTCGACAATGAGAAGGAGGCACAGATCTTGC GCGGATCCTCAACATAAGCAACCACTGAG

BiFC-YN-DEP1Δ79 GGGGACAAGTTTGTACAAAAAAGCAGGC TTCATGGTGAGCAAGGGCGAGG GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAACATAAGCAACCACTGAG

BiFC-RGB1-1 GTCGACAATGGCGTCCGTGGCGGAGCTCAAGG GGATCCTCAAACTATTTTCCGGTGTC

BiFC-YN-RGB1 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGTGAGCAAGGGCGAGG GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAAACTATTTTCCGGTGTCC

BiFC-YC-RGB1 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGACAAGCAGAAGAAC GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAAACTATTTTCCGGTGTCC

BiFC-RGA1-1 CGGTCGACAATGTCCGTGCTTACCTGTGT GCGGATCCTCAAGTTCCTTCCCTGGAG

BiFC-YN-RGA1 GGGGACA AGTTTGTACAAAAAAGCAGGCTTCATGGTGAGCAAGGGCGAGG GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAAGTTCCTTCCCTGGAGC

BiFC-YC-RGA1 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGACAAGCAGAAGAAC GGGGACCACTTT GTACAAGAA AGCTGGGTCTCAAGTTCCTTCCCTGGAGC

BiFC-RGG1-1 GTCGACAATGCAGGCCGGAGGAGGAGG ACTAGTTCACAAAAACCAGCATTTGCA

BiFC-YN-RGG1 GGGGACA AGTTTGTACAAAAAAGCAGGCTTCATGGTGAGCAAGGGCGAGG GGGGACCACTTT GTACAAGAAAGCTGGGTCTCACAAAAACCAGCATTTGC

BiFC-YC-RGG1 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGACAAGCAGAAGAAC GGGGACCACTTT GTACAAGAAAGCTGGGTCTCACAAAAACCAGCATTTGC

BiFC-RGG2-1 GCGTCGACAATGAGGGGGGAGGCGAACGG GCGGATCCCTAGGAAAAATCTGAGCCT

BiFC-YN-RGG2 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGTGAGCAAGGGCGAGG GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGGAAAAATCTGAGCCT

BiFC-YC-RGG2 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGACAAGCAGAAGAAC GGGG ACCACTTTGTACAAGAAAGCTGGGTCCTAGGAAAAATCTGAGCCT

AD-RGA1 GCCGAATTCATGTCCGTGCTTACCTGTG GCGGATCCTCAAGTTCCTTCCCTGGAG

BD-RGB1 CCCGGGTATGGCGTCCGTGGCGGAGCT GGATCCTCAAACTATTTTCCGGTGTC


AD-RGG1 GCGAATTCATGCAGGCCGGAGGAGGAGG GCGTCGACTCACAAAAACCAGCATTTGC

AD-RGG2 GCGAATTCATGAGGGGGGAGGCGAACGG GCGGATCCCTAGGAAAAATCTGAGCCT

AD-DEP1 CGGAATTCATGGGGGAGGAGGCGGTGGTG GCGGATCCTCAACATAAGCAACCACTGAG

AD-dep1 CGGAATTCATGGGGGAGGAGGCGGTGGTG GCGGATCCCTAGATGTTGAAGCAGGTGCAG

AD-DEP1ΔC1 CGGAATTCATGGGGGAGGAGGCGGTGGTG CGGGATCCTCAGCAGTGTCCCTCGCAGCA

AD-DEP1ΔC2 CGGAATTCATGGGGGAGGAGGCGGTGGTG CGGGATCCTCAGGAGCATTTGAAGATG

AD-DEP1ΔC3 CGGAATTCATGGGGGAGGAGGCGGTGGTG CGGGATTCTCAGCAGCTTGGAAGGCCACAG

AD-DEP1ΔC5 CGGAATTCATGGGGGAGGAGGCGGTGGTG CGGGATCCTCAGTTAGGTTTACAGCATGA

AD-DEP1ΔC6 CGGAATTCATGGGGGAGGAGGCGGTGGTG CGGGATCCTCACTTTGTTGGTATTAGTGG

AD-DEP1ΔN1 CGGAATTCATGAGAAGGAGGCACAGATCTTGC GCGGATCCTCAACATAAGCAACCACTGAG

AD-DEP1ΔN2 CGGAATTCATG TGCAGTTGCTGCAAGACCCCT GCGGATCCTCAACATAAGCAACCACTGAG

AD-DEP1ΔN3 GCGAATTCATGATCTTTTCATGCTTCAAATC GCGGATCCTCAACATAAGCAACCACTGAG

AD-DEP1ΔN4 CGGAATTCATGGGTTGCAACGGCTGCGGC TCG GCGGATCCTCAACATAAGCAACCACTGAG

AD-DEP1ΔN5 CGGAATTCATGTGCGCTGGCTGCTGCTCGAGC GCGGATCCTCAACATAAGCAACCACTGAG

AD-DEP1Δ56-63 CGGAATTCATGGGGGAGGAGGCGGTGGT CCTCCTTCTCTTTGT ATGTTTTGTACCAACAAACT


AD-DEP1Δ56-72 CGGAATTCATGGGGGAGGAGGCGGTGGT TGGTATTAGTGGGTCAACGGGCTGAGCTCCTTCAAG

GGAGCTCAGCCCGTTGACCCACTAATACCAACAAA GCGGATCCTCAACATAAGCAACCACTGAG

AD-DEP1Δ56-79 CGGAATTCATGGGGGAGGAGGCGGTGGT AGAAGGAGGCACAGAAACGGGCTGAGCTCCTTCAAG

GGAGCTCAGCCCGTTAGAAGGAGGCACAGATCTTG GCGGATCCTCAACATAAGCAACCACTGAG

AD-DEP1 1-56 CGGAATTCATGGGGGAGGAGGCGGTGGT CGGGATCCTCAAGAAACGGGCTGAGCTC

AD-DEP1 1-72 CGGAATTCATGGGGGAGGAGGCGGTGGT GCGGATCCTCAATGTTTTGTACCAACAAACT


Supplementary Table 5. Primer sequences used for transcript analysis.

Primers Forward sequence(5-3) Reverse sequence(5-3)

Actin3 CCACTATGTTCCCTGGCATT GTACTCAGCCTTGGCAATCC

AMT1;1 GGTTTCTCTCCCTCTCCGAT CCACCTTCACACCACACATT

AMT1;2 AAGCACATGCCGCAGACA GACGCCCGACTTGAACAG

AMT1;3 GAACGCGACGGACTACC CTGTGGGACCTGCTTGAG

GS1;1 CACCAACAAGAGGCACAATG ACTCCCACTGTCCTGGCAT

GS1;2 TGTTTCTCCTCATCCCTGC TCACAGTCCTCGCTTTGC

NADH-GOGAT1 GTGCAGCCTGTTGCAGCATAAA CGGCATTTCACCATGCAAATC

NADH-GOGAT2 CCTGTCGAAGGATGATGAAGGTGAAACC TGCATGGCCCTACTATCTTCGCATCA

Fd-GOGAT GCATACTTGTGAAGCACCGAAGTG CTGCAAATAGCAACCTAGCGTCAG

GDH1 CATCTGATCATCTCCCTGTT TTCAGGCAATTCATCACTAC

NiR CGAGGAGTAGGAACACAG TGTCGTCTACTTTACAAGGA

As TTACCTAAGCACATTCTATACAG CCTTCAATCCATCAATCCAA

NR CCTGGAGAAGATGGGCTAT GCACAACCATCCATCAATC

DEP1/dep1 GCGAGATCACGTTCCTCAAG TGCAGTTTGGCTTACAGCAT

RGB1 TGCCTCACAAGATGGAAG GAGTTAAGATTGAAGATAGAGC

CYCD3 CCTTCCACACTGACGGTACAGTT TGCCGCTGCCAAATAGACA

CYCD4 GCCATGGAGTTGATACATCCAA CCAGTAGGGCTCCGTGGAAT

CAK1 GACGGTCAGATTAGACGCAAGA TCCAAAGGATGTCCACA

CAK1A GACCGACAAGGGTTTCAGCAT CCAGCATGTTCAGGAAGATACAAT

CDKA1 GGTTTGGACCTTCTCTCTAAAATGC AGAGCCTGTCTAGCTGTGATCCTT

CDKA2 CGAGATTTGAAGCCCCAGAA TCCGCGAGCTTCAATGAGTT

CYCT1 GCATTTGTTGCAGCTCAAG TCACCACTTCGCTGACTTATTG

E2F2 TGTTGGTGGCTGCCGATAT CGCCAGGTGCACCCTTT

H1 GCAAGGCACCTGCAGCTT AGGCAGCCTTTGTACAGATCCT

MCM2 AAGTTGGCAAAAGATCCACGG CCCCCAAACATAGCTAGTGCAA

MCM3 TTCATGCGTCACTAAATGCGAG TGAATCTGGAAGCCCAATGTTC

MCM4 CCCGAATGCGATTCTCTGAA ACCAGTGGCATGATCAGTTGC

MCM5 AAGGAGAACTGCCTGTCCATGA AGTGGCCTTAGCTTTCACCCTC

CDT2 AACCGCACCAAACACTGGAA GCAATTCACCATCTGCACTGG

CYCA2.1 AGGTTGTCAAGATGGAGAGCGA CGCTTTTTGTCTTCCTGGCA

CYCA2.2 AGGTTGTCAAGATGGAGAGCGA CGCTTTTTGTCTTCCTGGCA

CYCA2.3 GTTTCGGTTGACGAGACGATGT CGCTGCAAGGAACCTAGAACTG

CYCB2.1 AAGTTTGGCCAGGAGTGAGCA TCAAGAGCATCAGCGTCGAGA

CYCB2.2 CTCAAGGCTGCACAATCTGACA GCATTGACGGCTGGAATTTG

CYCIaZm CACTCTCAAGCACCACACTGGA ACAACCCTCAGCTTGCTCTCAG

CDKB AAGTTTGGCCAGGAGTGAGCA TCAAGAGCATCAGCGTCGAGA

MAPK ACAGAGCAGCCGAATTTTGAGA TTCAGCGAAGCTCACACTTGG

KN CACCAGCTTCAAGAGATCGTGA CCGGAATTGAGACACAACTGC

CDC20 TCGAATCACCTGTTTGTTGGC TGGAGACAATCCAACGCAAAG


MAD2 GAGCCATGCATATTCGACGTG GGTGTCGAAGGAATGCAGCTT

RGA1 GCTACACATCAGTTATCCAT CAACCTGCCATCAATATCT

RGB1 GGGTTCATGTGATGCAACTG CCCCTCATGAGAGTTTTGGA

RGG1 ATAAGGTGTCAGCAGCATT TAGTCACTGGAAGTAGAGGAT

RGG2 CTTCTTCCTGTCACCATT CATTTGTTACTGCGAGAG

DEP1 CCGTTTCTCGTTCTGGAT ATCTGTGCCTCCTTCTCT


Supplementary Figures

Supplementary Figure 1 Map-based cloning of qNGR9. (a) The candidate

gene was mapped to the 18.6-kb region on rice chromosome 9, which was

defined by molecular markers W13 and W18. The number below the line

indicates the number of recombinants between qNGR9 and each marker. (b)

Polymorphisms of Os09g0441700 between QZL2 and NJ6. (c) Polymorphisms

of Os09g0441900 between QZL2 and NJ6.


Supplementary Figure 2 The genetic complementation. (a) NIL-qNGR9

plants exhibited a marked nitrogen response with respect to plant height. (b)

NIL-qNGR9 plants over-expressing dep1-1 cDNA amplified from QZL2 showed

nitrogen-insensitive responses. (c) NIL-dep1-1 plants are largely unresponsive to

nitrogen input level. Scale bar: 25 cm.


Supplementary Figure 3 The expression pattern of dep1-1. Transcriptional

level of dep1-1 was induced by the availability of nitrogen. Transcript abundance

relative to the level of 2-month-old NIL-dep1-1 plants grown without nitrogen

fertilization set to be one. Data shown as mean ± SE (n = 3).


Supplementary Figure 4 Comparisons of culm length and leaf width

between NIL-DEP1 and NIL-dep1-32 plants at the mature stage. (a) The

culm length. Scale bar: 10 cm. (b) Phenotype of flag leaves. Scale bar: 3 cm. (c,

d) The epidermal cell morphology of leaf blades.


Supplementary Figure 5 The dep1-1 and dep1-32 alleles exhibit insensitive

growth to nitrogen input level. 21-day-old seedlings exposed to a hydroponic

solution containing either low (0.035 mM) or high (1.4 mM) concentrations of

NH4NO3.


Supplementary Figure 6 Genetic diversity analysis at the DEP1 locus. The blue and red dots indicate the Fst (Fixation index) values of all

site nucleotide diversities in a ~4.7 kb region in chromosome 9 (17117300-1712200, IRGSP genome sequence build 5) encompassing the entire

DEP1 sequence in O. rufipogon and indica relative to japonica, respectively. The vertical bars showed that the three non-synonymous SNPs in

the CDS region of DEP1, which resulted in three FNPs (p.105C＞Y, p.228H＞L and p.324S＞C), clearly differentiate the japonica cultivar and

O. rufipogon.


Supplementary Figure 7 The effect of the dep1-1 allele on cell proliferation.

Longitudinal sections of the uppermost internode of the NIL plants shown in Fig.

1h. Scale bar: 0.1 mm.

Supplementary Figure 8 Effect of the dep1-1 allele on the expression of genes

involved in determining cell cycle time. Transcriptional level was determined

by quantitative real time PCR based on cDNA template prepared from young

internodal tissues. Rice actin3 served as the reference gene. Data shown as mean

± SE (n = 12). The values were expressed relative to the level of transcript in

NIL-DEP1 set to be one.


Supplementary Figure 9 The transcription of genes involved in nitrogen

uptake and assimilation in the roots. Seedlings were exposed for 14 days to a

hydroponic solution containing either low (0.035 mM) or high (1.4 mM)

concentrations of NH4NO3. Transcript level expressed as the number of transcript

copies per 1000 copies of rice actin3. Data shown as mean ± SE (n = 6).


Supplementary Figure 10 The transcription of genes involved in nitrogen

uptake and assimilation in the leaves. Seedlings were exposed for 14 days to a


concentrations of NH4NO3. Transcript level expressed as the number of transcript

copies per 1000 copies of rice actin3. Data shown as mean ± SE (n = 6).


Supplementary Figure 11 Effects of nitrogen input level on expression of rice

GLRs. The NIL-DEP1 and NIL-dep1-1 plants were exposed for 28 days to a


concentrations of NH4NO3. Relative expression shown as the number of

transcript copies per 1000 copies of rice actin3. Data shown as mean ± SE (n =

3). Transcript level of the rice glutamate-like receptor homologs (GLRs) in

NIL-dep1-1 was up-regulated: Os06g09090 (GLR2.8), Os04g49570 (GLR3.1),

Os02g02540 (GLR3.3), Os07g33790 (GLR3.4) and Os06g46670 (GLR3.4-like).


Supplementary Figure 12 Comparison of glutamine synthase activity

between NIL-dep1-1 and NIL-DEP1 plants. (a) 14 day old seedlings exposed

to either low (0.035 mM) or high (1.4 mM) concentrations of NH4NO3. (b) 4

week old leaf. The paddy-grown rice plants raised as described in Fig. 2i. Data

shown as mean ± SE (n = 6). Student’s t-test was used to generate the P values. *,

highly significantly different (P < 0.01); ns, not significant (P ＞0.05).


Supplementary Figure 13 Comparison of plant architecture between NIL

plants grown in low and high nitrogen conditions. (a) The number of tillers

per plant. (b) Plant height. (c) The number of grains per panicle. Data shown as

mean ± SE (n = 30). The paddy-grown rice plants under the two contrasting

nitrogen input levels (LN: low N, 60 kg/ha; HN: high N, 300 kg/ha). The

presence of the same lower case letter above histogram bars denotes

non-significant differences (P < 0.05).


Supplementary Figure 14 Effects of the variant alleles of DEP1 on harvest

index (HI). (a) Comparison of HI between NIL-DEP1 and NIL-dep1-1 plants. (b)

Comparison of HI between NIL-dep1-1 and NIL-sd1 plants. Measurements made

from 40 plants raised as described in Fig. 2k. Data shown as mean ± SE (n = 6).


Supplementary Figure 15 The effect of the rice G-proteins on plant height.

(a) Sub-cellular localization of the dep1-1-GFP fusion protein in the roots of

transgenic rice plants. (b) The phenotype of transgenic plants carrying

pActin::myc-DEP1. Scale bar: 15 cm. (c) The morphology of transgenic cv.

Nipponbare over-expressing RGA1 at the mature stage. Scale bar: 15 cm. (d) The

transgenic Nipponbare plants over-expressing the canonical γ-subunits (RGG1

and RGG2) were dwarfed. Scale bar: 20 cm. (e) The phenotype of the transgenic

Nipponbare plants over-expressing GS3. Scale bar: 20 cm.


Supplementary Figure 16 Identification of a new variant at the d1 locus. (a)

The Chinese cultivar Xueheaizao (XHAZ) is a semi-dwarf. Scale bar: 15 cm. (b)

Allelic variation at RGA1 (LOC_Os05g26890) in the d1XHAZ mutant. (c) The

transcription of RGA1 in NIL-D1 and NIL-d1XHAZ plants. Transcript abundance

normalized against that measured in NIL-D1. Data shown as mean ± SE (n = 3).


Supplementary Figure 17 The nitrogen response associated with allelic

variants of DEP1. (a) Genetic diversity analysis at the CDS region of DEP1.

The blue and red circles indicate the Fst (Fixation index) values of all site

nucleotide diversities in O. rufipogon and indica relative to japonica,

respectively. The vertical bars showed that the clearly differentiate

non-synonymous SNPs in the ORF region (c.314G＞A, c.683A＞T and c.970A

＞T) between O. rufipogon and the japonica cultivar. (b) Yeast two-hybrid

assays. The deleted versions of DEP1 as described in Fig. 3b. Data shown as

mean ± SE (n = 3). The presence of the same lower case letter above histogram

bars denotes non-significant differences (P < 0.05).


Supplementary Figure 18 The phenotypes of RGB1 over-expressors grown

in the low nitrogen conditions. (a) RGB1 transcript abundance in four

independent transgenic lines (transcript abundance normalized against that

measured in NIL-DEP1). (b) Tiller number developed by the transgenic line T9

(see a) and non-transgenic NIL-DEP1 plants grown under either low (30 kg/ha)

or high (300 kg/ha) nitrogen availability. Data shown as mean ± SE (n = 20).

Student’s t-test was used to generate the P values. *, significantly different

(P<0.05); ns, not significant (P ＞ 0.05). (c) Flag leaf morphology of

paddy-grown transgenic plants grown under either low (30 kg/ha) or high (300

kg/ha) nitrogen availability. (d) 21-day-old seedlings exposed to either low

(0.035 mM) or high (1.4 mM) concentrations of NH4NO3.


qngr9 and genetic complementation. · supplementary note: map-based cloning of qngr9 and genetic...

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