Functional conservation and divergence of GmCHLI genes inpolyploid soybean

Qing Li1,2, Chao Fang1,2, Zongbiao Duan1,2, Yucheng Liu1,2, Hao Qin3, Jixiang Zhang1,2, Peng Sun4, Wenbin Li5,

Guodong Wang3 and Zhixi Tian1,*1State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese

Academy of Sciences, Beijing 100101, China,2University of Chinese Academy of Sciences, Beijing 100039, China,3State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,

Beijing 100101, China,4Affiliated Hospital of Hebei University, Baoding 071000, China, and5Key Laboratory of Soybean Biology in Chinese Ministry of Education, Northeast Agricultural University, Harbin 150030,

China

Received 20 June 2016; revised 17 July 2016; accepted 21 July 2016.

*For correspondence (e-mail zxtian@genetics.ac.cn).

Accession numbers: RNA-seq raw sequence data generated from this study have been deposited in the Genome Sequence Archive (GSA) database in the BIG

Data Center (http://gsa.big.ac.cn/index.jsp) under accession number PRJCA000242.

SUMMARY

Polyploidy is prevalent in nature. As the fate of duplicated genes becomes more complicated when the

encoded proteins function as oligomers, functional investigations into duplicated oligomer-encoding genes

in polyploid genomes will facilitate our understanding of how traits are expressed. In this study, we identi-

fied GmCHLI1, a gene encoding the I subunit of magnesium (Mg)-chelatase, which functions in hexamers as

responsible for the semi-dominant etiolation phenotype in soybean. Four GmCHLI copies derived from two

polyploidy events were identified in the soybean genome. Further investigation with regard to expression

patterns indicated that these four copies have diverged into two pairs; mutation in the other copy of the

pair that includes GmCHLI1 also resulted in a chlorophyll-deficient phenotype. Protein interaction assays

showed that these four GmCHLIs can interact with each other, but stronger interactions were found with

mutated subunits. The results indicate that, in polyploidy, deficiency in each copy of duplicated oligomer-

encoding genes could result in a mutant phenotype due to hetero-oligomer formation, which is different

from the model of allelic dosage or functional redundancy. In addition, we interestingly found an increase in

isoflavonoids in the heterozygous etiolated plants, which might be useful for improving soybean seed

quality.

Keywords: duplicated genes, GmCHLIs, oligomers, evolution, soybean.

INTRODUCTION

Polyploidy, or whole-genome duplication (WGD), is com-

mon in nature, particularly in plants (Wendel, 2000; Kon-

drashov et al., 2002), and the duplicated genes generated

through polyploidy provide important raw genetic material

for evolution (Zhang, 2003; Conant and Wolfe, 2008). Inves-

tigations of the fate of duplicated genes will help our

understanding of allelic divergence, novel phenotypic gen-

eration and adaptive evolution (Conant and Wolfe, 2008).

Since the first report of gene duplication in Drosophila in

1936 (Bridges, 1936), duplicated genes have been exten-

sively studied at phylogenetic, functional and genomic

levels (Adams and Wendel, 2005; Innan and Kondrashov,

2010). During evolution, one copy of a duplicated pair may

be lost, leaving the other copy as a singleton. However,

when both copies of a duplicated pair persist, the two

copies will generally evolve with different fates and can

maintain all or only part of their original functions, exhibit

divergent functions and gain new functions; one or both

copies can also become pseudogenes (Flagel and Wendel,

2009).

Divergence between duplicate genes can arise via diver-

gence of their expression patterns or via accumulated

© 2016 The AuthorsThe Plant Journal © 2016 John Wiley & Sons Ltd

1

The Plant Journal (2016) doi: 10.1111/tpj.13282

http://gsa.big.ac.cn/index.jsp

mutations (Innan and Kondrashov, 2010). Studies have

indicated that sub-functionalization at the expression level

might be a relatively rapid process compared with sub-

functionalization through mutations (Doyle et al., 2008;

Roulin et al., 2012). One example is soybean, an important

crop providing major sources of animal feed protein and

cooking oil. It has been reported that the soybean genome

has undergone two polyploidy events during the last 60

million years (Shoemaker et al., 2006), which have resulted

in approximately 75% of its genes being present in more

than one copy (Schmutz et al., 2010). Additionally, gen-

ome-wide expression profiling has demonstrated that

approximately 50% of the paralogs in soybean are differen-

tially expressed (Roulin et al., 2012). For instance, only one

copy of the duplicated genes homologous to Arabidopsis

TERMINAL FLOWER controls growth habit in soybean; the

other copy does not due to a divergent transcriptional

pattern (Tian et al., 2010).

In addition to evolving into divergent functional pairs,

some duplicated pairs maintain a similar function (Zhang,

2003). Investigations of the coordinated functions of these

conserved duplicated copies are interesting and will help

to clarify how a trait is determined, which is important for

crops because almost all crop plants are polyploid (Udall

and Wendel, 2006). One popular model of conserved dupli-

cated gene cooperation is the control of a trait by allelic

dosage. For instance, studies indicate that the different

copies of FLOWERING LOCUS C (FLC) in Brassica napus, a

polyploid crop, affect flowering time incrementally, which

explains a large portion of the phenotypic variance

observed in field trials (Schranz et al., 2002; Quijada et al.,

2006; Udall et al., 2006). Another popular model in trait

control is functional redundancy. For example, D1 and D2,

homologs of the STAY-GREEN (SGR) gene, are duplicated

as a result of the most recent WGD in soybean: only the

d1d2 double mutant was found to exhibit the stay-green

phenotype, whereas the single mutant did not, suggesting

that D1 and D2 are redundantly involved in chlorophyll

degradation (Fang et al., 2014).

In nature, as opposed to acting directly as monomers,

many proteins must assemble as oligomers to perform

their functions. Following polyploidy, hetero-oligomers

instead of homo-oligomers may form. Thus, the evolution

of conserved duplicated genes becomes more complicated

when their encoded proteins function as oligomers. Func-

tional and evolutionary investigations of these duplicated

oligomer-encoding genes in polyploid genomes will pro-

vide important information regarding how traits are

expressed.

Magnesium (Mg)-chelatase, which catalyzes the first

step of chlorophyll biosynthesis in a heterotrimeric com-

plex, is composed of three distinct subunits in plants: CHLI,

CHLD and CHLH (Rissler et al., 2002). Structural analyses

have suggested that CHLI forms hexamers and belongs to

the family of ATPases associated with various cellular

activities (AAA+) (Fodje et al., 2001; Hansson et al., 2002).

In barley and maize, CHLI is encoded by a single gene, and

mutation of this gene results in the semi-dominant chlo-

rina phenotype (Hansson et al., 1999; Sawers et al., 2006).

Most genes in soybean are present in multiple copies due

to the WGDs that have occurred (Schmutz et al., 2010). In

this study, we performed map-based cloning and found

that a CHLI homolog in soybean, GmCHLI1, is responsible

for the etiolation phenotype. Four paralogous copies are

presented in the soybean genome, and we comprehen-

sively investigated functional conservation and divergence

among these four copies. We found that the duplication

status of soybean CHLIs has greatly influenced their role

in chlorophyll biosynthesis due to the formation of

hetero-oligomers.

RESULTS

GmCHLI is the gene responsible for the etiolation

phenotype in soybean

We found that one soybean accession in our germplasm

pool always exhibited segregating phenotypes in the pro-

geny population, including green, pale-green, and yellow

leaves (Figure 1a). Further investigation revealed lethality

of the yellow plants shortly after germination and that the

green plants did not generate segregating progeny. How-

ever, the progeny derived from the pale-green plants

exhibited a semi-dominant segregation ratio of 1:2:1 for

green:pale-green:yellow phenotypes. Therefore, we deter-

mined the etiolation phenotype (termed the y mutant in

this study) to be controlled by a single semi-dominate

gene. The green, pale-green and yellow plants were of the

Y/Y, Y/y and y/y genotypes, respectively.

Consistent with their phenotypes, the y/y unifoliolate

leaf contained much smaller chlorophyll (both Chl a and

Chl b) and carotenoid contents than those of the Y/Y unifo-

liolate leaf (Figure 1b). Ultrastructural investigation by

transmission electron microscopy suggested that the

chloroplasts of the mutant were also affected (Figure 1c).

Compared with the chloroplasts of Y/Y unifoliolate leaf, the

chloroplasts of y/y unifoliolate leaf showed abnormal

structures, with no clear prolamellar body and only a few

prothylakoid membranes extending across the length of

the chloroplasts (Figure 1c). To determine whether photo-

synthesis efficiency was affected in the mutant, the chloro-

phyll fluorescence parameters Fv/Fm (maximal PS II

quantum yield), NPQ (nonphotochemical quenching) and

qP (coefficient of photochemical quenching) were mea-

sured. The Fv/Fm, NPQ and qP values of the y/y unifolio-

late leaf were significantly decreased compared with those

of the Y/Y unifoliolate leaf (Figure 1d–f). Interestingly,although the Y/y unifoliolate leaf exhibited intermediate

deficiency in chlorophyll and carotenoid levels (Figure 1b)

© 2016 The AuthorsThe Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), doi: 10.1111/tpj.13282

2 Qing Li et al.

and chloroplast structures (Figure 1c), its chlorophyll fluo-

rescence parameters were similar to those of the Y/Y unifo-

liolate leaf (Figure 1d–f). This situation indicates thatalthough chlorophyll deficiency occurs in Y/y plants, the

chlorophyll level is still above the threshold for maintain-

ing normal photosynthesis.

To identify the y locus, we crossed the Y/y mutant with

Williams 82. Initial mapping using an F2 population con-

sisting of approximately 350 individuals anchored the y

locus in a 0.22 Mb region between ‘CAPS marker 25’ and

‘CAPS marker 23’ on chromosome 13 (Figure 1g). In total,

37 annotated genes are present in this 0.22 Mb region.

Except for Glyma13g30560, the annotated functions of

these genes are either unknown or do not involve chloro-

phyll metabolism (Table S1). Glyma13g30560 encodes a

CHLI homolog, the I subunit of Mg-chelatase, and CHLI

mutation results in a semi-dominant chlorophyll-deficient

phenotype in various plant species (Figure S1) (Hansson

et al., 1999; Sawers et al., 2006; Huang and Li, 2009).

Comparison with the Y/Y genomic DNA sequence demon-

strated four SNPs in y/y plants (Figure 1g). One SNP is a

non-synonymous A->G mutation in the third exon, whichconverts a Gln to an Arg at residue 275; the remaining

SNPs are synonymous mutations in non-coding regions.

The Y/y plants were found to be heterozygous at these

positions. Thus, we speculated that Glyma13g30560 is a

candidate gene responsible for the semi-dominant etiola-

tion phenotype of the y mutant and that A->G is theresponsible mutation. This speculation was confirmed by

a recent study, in which the same missense mutation in

GmCHLI was identified in an investigation of a semi-

dominant chlorophyll-deficient mutant of y11 through a

candidate gene sequencing method (Campbell et al.,

2015).

Y/Y Y/y y/y

Y/Y Y/y y/y

Y/Y Y/y y/y

1 µm

Fv/F

m

NP

Q

qP

Pig

men

ts (m

g·g-

1 fre

shw

eigh

t)

0

0.2

0.4

0.6

0.8

1

0

1

2

3

4

5 Chl aChl bCar

0

0.2

0.4

0.6

0.8

0

0.1

0.2

0.3

0.4

0.5

b a c

a a

b

aa

b

(a) (b)

(c)

(d) (e) (f)

(g)

Recombinants

Chr. 13

4 Mb

32.5

4 M

b34

.39

Mb

100 kb

CA

PS

28

CA

PS

22

3 2 1 110 0 1 1 2 3 7 15

Candidate region223 kb

10 kb

500 bp

5735 bpGlyma13g30560

UTR Exon

ATG TGAG->A

A->G A->TC->T

y

1 cm

1 µm1 µm

Y/Y Y/y y/y Y/Y Y/y y/y Y/Y Y/y y/y

Figure 1. Identification of the y locus.

(a) Fourteen-day-old Y/Y, Y/y and y/y seedlings.

(b) Chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoid (Car) contents in unifoliolate leaf, as shown in (a). The error bar indicates the standard deviation

of three replicates.

(c) Ultrastructure of the chloroplasts in unifoliolate leaf, as shown in (a).

(d–f) Chlorophyll fluorescence values of Fv/Fm (d), NPQ (e) and qP (f) in unifoliolate leaf, as shown in (a). The error bar indicates the standard deviation of atleast three replicates. Statistical significance at the 5% level was determined using Duncan’s multiple range test.

(g) Map-based cloning of the y locus.

© 2016 The AuthorsThe Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), doi: 10.1111/tpj.13282

Evolution of GmCHLIs in soybean 3

Four GmCHLI copies are present in the soybean genome

Soybean is a paleopolyploid plant that has experienced at

least two rounds of WGD events (Shoemaker et al., 2006),

resulting in approximately 75% of its genes being present

in multiple copies (Schmutz et al., 2010). A homolog

search showed four CHLI copies in the soybean genome

(Figures 2a and S1). To distinguish them from each other,

we termed Glyma13g30560 as GmCHLI1 and the other

three copies as GmCHLI2 (Glyma15g08680), GmCHLI3 (Gly-

ma07g32550) and GmCHLI4 (Glyma13g24050). These four

copies were further grouped into two pairs: GmCHLI1 and

GmCHLI2; GmCHLI3 and GmCHLI4 (Figure 2a). The

sequence similarity within each pair was higher than that

between the pairs (Figure 2a and Table S2). Based on a

previous WGD analysis (Schmutz et al., 2010), we deter-

mined that the copies within each pair came from the

younger WGD at 13 million years ago (MYA) and that the

two pairs were derived from the older WGD at 59 MYA

(Figure S2).

The existence of duplicated genes provides opportuni-

ties for functional divergence (Force et al., 1999; Lynch

et al., 2001), and it would be valuable to determine how

the four GmCHLI copies coordinately evolved to control

chlorophyll biosynthesis. An expression assay using previ-

ously reported transcriptome data (Shen et al., 2014) sug-

gested that GmCHLI1 and GmCHLI2 exhibit significantly

different expression patterns from those of GmCHLI3 and

GmCHLI4 (Figure 2b). Consistent with their function in

photosynthesis, GmCHLI1 and GmCHLI2 exhibited con-

stantly high expression in most tissues, except for the root,

senescent leaf and later-developed seed. In contrast,

GmCHLI3 and GmCHLI4 exhibited significantly lower

expression in most tissues and presented only relatively

higher expression in young tissues (Figure 2b). These

(a)

(b)

(c)

(d)

(e)

AtCHLI2

HvCHLI78

100

100

100

99

99

90

0.02

GmCHLI1GmCHLI2

GmCHLI3GmCHLI4

AtCHLI1

NtCHLIZmCHLI

OsCHLI

LUC

/RE

N

0

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

PAtCHLI1 PAtCHLI2 PGmCHLI1 PGmCHLI2

PGmCHLI3 PGmCHLI4 pGreen CK

GmCHLI1

GmCHLI2

GmCHLI3

GmCHLI4

LB RB

KpnI/XhoI SpeIPromoter

REN LUC35S

Roo

tS

tem

-1S

tem

-2S

hoot

mer

iste

mC

otyl

edon

-1C

otyl

edon

-2Le

af b

ud-1

Leaf

bud

-2Le

af b

ud-3

Leaf

-1Le

af-2

Leaf

-3Fl

ower

-1Fl

ower

-2Fl

ower

-3Fl

ower

-4Fl

ower

-5P

od+s

eed-

1P

od+s

eed-

2P

od+s

eed-

3P

od-1

Pod

-2P

od-3

See

d-1

See

d-2

See

d-3

See

d-4

See

d-5

Figure 2. Phylogenetic analysis and transcriptional assay of different GmCHLI copies.

(a) Phylogenetic analysis of CHLI homologs/paralogs in soybean and other plants. A neighbor-joining tree was constructed with MEGA 6.0 software based on

amino acid sequences. The protein sequences are from soybean (GmCHLI1, Glyma13 g30560; GmCHLI2, Glyma15 g08680; GmCHLI3, Glyma07 g32550 and

GmCHLI4, Glyma13 g24050), Arabidopsis (AtCHLI1, At4 g18480; AtCHLI2, At5 g45930), Oryza sativa (OsCHLI, Os03 g36540.1), Zea mays (ZmCHLI,

GRMZM2G419806_T01), Hordeum vulgare (HvCHLI, ABF72535.2) and Nicotiana tabacum (NtCHLI, AAG35472.1).

(b) Transcriptional assay of the four GmCHLI copies in 28 tissues/organs. The size of the number indicates earlier to later developmental stages within the same

tissue/organ. The FPKM value was recognized as the gene expression level; the data are shown in base 2 logarithmic form (log2 FPKM).

(c) Diagram showing the structure of the Dual-Luciferase reporter vector.

(d) LUC activities driven by the indicated CHLI promoters in N. benthamiana.

(e) Dual-Luciferase assay of the relative CHLI promoter activity. The error bar indicates the standard deviation of three replicates.

© 2016 The AuthorsThe Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), doi: 10.1111/tpj.13282

4 Qing Li et al.

results suggest that the two pairs (GmCHLI1 and GmCHLI2;

GmCHLI3 and GmCHLI4) have evolved differential func-

tions; GmCHLI3 and GmCHLI4 may no longer be the major

genes controlling chlorophyll biosynthesis in soybean or at

least not in all tissues.

To investigate associations between differential tran-

scription levels and promoter divergence, we assessed the

promoter activities of the four GmCHLIs using a reported

transient transcription Dual-Luciferase assay system (Hel-

lens et al., 2005) in which tobacco leaves were infected

with Agrobacterium tumefaciens harboring one plasmid

expressing a Dual-Luciferase reporter (Figure 2c, d). In Ara-

bidopsis, CHLI is encoded by two copies, AtCHLI1 and

AtCHLI2 (Rissler et al., 2002), with AtCHLI1 being

expressed at a much higher level than AtCHLI2 and thus

acting as the major functional gene (Huang and Li, 2009).

To validate the reliability of our experimental system, the

promoter activities of AtCHLI1 and AtCHLI2 were also

quantified as positive controls. The results showed that the

transcriptional activity (ratio of LUC to REN) of PAtCHLI1

was approximately six times higher than that of PAtCHLI2

(Figure 2e), which is consistent with the mRNA ratio of

AtCHLI1 to AtCHLI2 (5.86 � 1.31) in Arabidopsis seedlings(Huang and Li, 2009) and demonstrated the reliability of

the experimental system. Consistent with their expression

levels, the promoters of GmCHLI1 and GmCHLI2 exhibited

significantly stronger transcription activities than the pro-

moters of GmCHLI3 and GmCHLI4 (Figure 2e). Our results

indicate that evolution of the promoter activities of the

different copies of GmCHLI may greatly affect their

transcription patterns.

Mutation of GmCHLI2 also results in a chlorophyll-

deficient phenotype

Analyses of expression and promoter activities suggested

that GmCHLI2 has characteristics similar to those of

GmCHLI1 (Figure 2b, e), indicating that GmCHLI2 may

have similar functions as GmCHLI1 with regard to chloro-

phyll biosynthesis. If so, mutation in GmCHLI2 may also

result in a chlorophyll-deficient phenotype. A previous

study reported that the y9 mutant resulted in a chlorina

foliage phenotype (Chen et al., 1999). y9 is located in the

region between the two markers ‘Satt720’ and ‘A069_2’ on

chromosome 15 (Cregan et al., 1999) (http://www.soy-

base.org/), and GmCHLI2 is also located in this interval.

Therefore, GmCHLI2 is a possible candidate for y9. Geno-

mic DNA sequencing of GmCHLI2 from the y9 mutant (PI

548172) and its parent Illini (PI 548348) showed a single

non-synonymous G->A mutation in the third exon, result-ing in the change of amino acid Asp to Asn at residue 278

(Figure 3a). The same amino acid substitution in HvCHLI

was reported for the barley Clo-125 mutant (Figure S1)

(Hansson et al., 1999). These results confirmed that

GmCHLI2 is a candidate for y9, a recessive mutant with

greenish yellow leaves (Figure 3b) and notably decreased

chlorophyll and carotenoid contents compared with Illini

(Figure 3c). Consistently, the grana stacks of y9 chloro-

plasts were looser than those of Illini (Figure 3d).

Functional analysis of the four GmCHLIs

The above analyses suggested that GmCHLI1 and

GmCHLI2 may have similar functions, and when duplicated

genes have similar functions, they usually exhibit func-

tional redundancy in trait control. Nevertheless, mutation

in either GmCHLI1 or GmCHLI2 could result in chlorophyll

biosynthesis deficiency. Moreover, although GmCHLI3 and

GmCHLI4 exhibited different expressional patterns com-

pared with GmCHLI1 and GmCHLI2, high similarities were

observed in the protein sequences of these four GmCHLIs

(Table S2). To clarify the functional divergence of these

GmCHLIs at the protein level, we first investigated their

subcellular localization. All four GmCHLIs were localized to

the chloroplasts, as is AtCHLI1, suggesting that their sub-

cellular localization had not diverged during evolution fol-

lowing the two WGD events. Moreover, we found that

neither mGmCHLI1 from the homozygous y mutant nor

mGmCHLI2 from the y9 mutant exhibited altered subcellu-

lar localization (Figure S3).

Subsequently, we performed a complementary test to

study the functions of these four GmCHLI copies. To

(a) GmCHLI27510 bp

UTR Exon 500 bp

ATG TGA

G->A

Illini y9

Illini y9

Illini y9

Pig

men

ts (m

g·g-

1 fre

shw

eigh

t)

0

1

2

3Car

Chl aChl b

1 µm

5 cm

y Q->R y9 D->N

(b) (c)

(d)

1 µm

Figure 3. Phenotypes of the y9 mutant.

(a) Mutation site in the GmCHLI2 gene and protein in the y9 mutant.

(b) Fourteen-day-old Illini and y9 seedlings.

(c) Chl a, Chl b and carotenoid (Car) contents in unifoliolate leaf, as shown

in (b). The error bar indicates the standard deviation of three replicates.

(d) Ultrastructure of chloroplasts in unifoliolate leaf, as shown in (b).

© 2016 The AuthorsThe Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), doi: 10.1111/tpj.13282

Evolution of GmCHLIs in soybean 5

http://www.soybase.org/http://www.soybase.org/

eliminate the potential influence of different GmCHLI

copies in the soybean genome, we used the Arabidopsis

cs215 mutant as the background because it has been

reported that CHLI is functionally conserved in various spe-

cies and only AtCHLI1 functions as the main I subunit of

Mg-chelatase in Arabidopsis (Huang and Li, 2009). cs215 is

a chli1 mutant with a non-synonymous mutation and pre-

sents a semi-dominant chlorophyll-deficient phenotype

(Huang and Li, 2009). The results showed that each of the

wild-type GmCHLIs driven by the 35S promoter could res-

cue the chlorophyll-deficient phenotype of the cs215

mutant, similar to the results for AtCHLI1 (Figure 4 and

Table S3). The transgenic plants presented foliage pheno-

types that ranged from yellow to fully green, and the

degree of rescue was generally positively related to the

level of transgenic expression (Figure S4a–d). In contrast,the mutant types of mGmCHLI1 and mGmCHLI2 could not

rescue the cs215 mutant (Table S3). Moreover, some

mGmCHLI1/mGmCHLI2 transgenic plants in the Enk

(wild-type) background were slightly green and even etio-

lated (Figure 4 and Table S3). Overall, the chlorina level

was also positively related to high transgenic expression

(Figure S4e, f). It is noteworthy that the chlorophyll-

deficient phenotype of the 35S::mGmCHLI2 transgenic

plants was not as serious as that of the 35S::mGmCHLI1

transgenic plants (Figure 4 and Table S3), which is consis-

tent with the corresponding phenotypes of the y and y9

mutants. These results indicate that all four GmCHLIs can

function as the CHLI subunit but that their functions could

be dominant negatively affected by non-synonymous

mutation of either copy of GmCHLI1 and GmCHLI2, further

suggesting that the different copies might interacted in

nature.

The four GmCHLIs interact with each other but exhibit

stronger interactions with mGmCHLI1 or mGmCHLI2

To reveal how these GmCHLIs cooperate to control of

chlorophyll synthesis, their interactions were evaluated

using a point-to-point yeast two-hybrid (Y2H) assay. All of

the combinations of wild-type GmCHLIs showed moderate

interactions at 6 days of incubation (Figure 5a). These

interactions were further confirmed using a bimolecular

fluorescence complementation (BIFC) assay (Figure S5).

Unexpectedly, the mutated subunits (mGmCHLI1 and

mGmCHLI2) not only showed interactions with the other

GmCHLIs, but the interactions were even slightly stronger

(Figure 5a). To validate this result, we examined the inter-

action capacities of the GmCHLI1 and GmCHLI2 with their

wild-type and mutated subunits in yeast cells using b-galassays (Figure 5b). The results showed that the mutated

subunits indeed exhibited stronger interactions compared

with the wild-type subunits, particularly between different

paralogs (GmCHLI1 with mGmCHLI2 and mGmCHLI1 with

GmCHLI2).

In addition to CHLI, CHLD and CHLH are also required

for the formation of a functional Mg-chelatase heterotri-

meric complex (Hansson et al., 2002; Rissler et al., 2002).

We found two CHLD copies (GmCHLD1 for Glyma01g43630

and GmCHLD2 for Glyma11g01840) and three CHLH copies

(GmCHLH1 for Glyma03g29330, GmCHLH2 for Gly-

ma10g20570 and GmCHLH3 for Glyma19g32070) in the

soybean genome. We determined that GmCHLD1 and

GmCHLD2 as well as GmCHLH1 and GmCHLH3 are

Enk cs215/+ cs215/cs215

35S::AtCHLI1 (cs215/cs215)

35S::GmCHLI1 (cs215/cs215)

35S::GmCHLI2 (cs215/cs215)

35S::GmCHLI3 (cs215/cs215)

35S::GmCHLI4 (cs215/cs215)

35S::mGmCHLI1 (Enk)

35S::mGmCHLI2 (Enk)

2 cm

Figure 4. Complementation tests of GmCHLIs in Arabidopsis.

Photographs of approximately 3-week-old Enk, cs215/+, cs215/cs215 and

representative transgenic lines are shown.

© 2016 The AuthorsThe Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), doi: 10.1111/tpj.13282

6 Qing Li et al.

duplication pairs from the recent WGD (Figure S2). Subcel-

lular localization showed that all of the GmCHLD and

GmCHLH copies localize to the chloroplast (Figure S3). We

then performed the BIFC assay to investigate interactions

between all four of the GmCHLIs and the different copies

of GmCHLD or GmCHLH, and the results indicated that

they all interacted (Figure S6). We also assessed interac-

tions between GmCHLDs and GmCHLHs, as well as the

interactions of GmCHLDs or GmCHLHs themselves, with

positive results for all combinations (Figure S7).

Expression of GmCHLIs, GmCHLDs and GmCHLHs are

affected in y and y9 mutants

Because all of the GmCHLIs, GmCHLDs and GmCHLHs

were found to interact with each other, mutation of

GmCHLI1 and GmCHLI2 might affect expression of these

subunits. We then explored the gene expression of these

subunits in the root, stem, cotyledon and unifoliolate leaf

of Y/Y, Y/y and y/y plants by RT-qPCR. First, the results

indicated much higher expression for GmCHLI1 and

GmCHLI2 relative to GmCHLI3 and GmCHLI4 in unifoliolate

leaf, which is consistent with the transcriptome results

(Figure 6a–d). GmCHLD1 showed a significantly higherexpression level than GmCHLD2 (Figure 6e, f), and the

mRNA levels of GmCHLH1 and GmCHLH3 were signifi-

cantly higher relative to that of GmCHLH2 (Figure 6g–i).Second, expect for GmCHLI4, all of the genes were highly

expressed in the unifoliolate leaf compared with other

tested tissues/organs (Figure 6). Third, expression of

almost all of the genes was decreased in the y/y unifolio-

late leaf in comparison with that of the Y/Y unifoliolate leaf,

with exception of GmCHLD2 (Figure 6). Consistent with the

phenotype (Figure 1), the total mRNA levels of these sub-

units in the Y/y unifoliolate leaf were comparable with

those in the Y/Y unifoliolate leaf, or at an intermediate level

between the Y/Y and y/y unifoliolate leaves (Figure 6). Sim-

ilarly, transcription of these subunits was evaluated in the

y9 and Illini plants (Figure S8), with expression in the y9

mutant either unchanged or slightly decreased in

comparison with Illini. These results indicate that the

GmCHLI1 and GmCHLI2 missense mutations not only

affect interaction capacity but also influence the transcript

abundance of Mg-chelatase subunits and subsequently

block chlorophyll biosynthesis.

Isoflavonoids are increased in Y/y plants

Isoflavonoids are special metabolites in legumes and also

one of the key parameters for evaluating the nutritional

quality of soybean seeds (Dhaubhadel et al., 2003). Isofla-

vonoids are synthesized via the general phenylpropanoid

pathway and legume-specific isoflavonoid branch path-

ways catalyzed by a series of enzymes, including pheny-

lalanine ammonia lyase (PAL), chalcone synthase (CHS),

chalcone reductase (CHR), isoflavone synthase (IFS), cinna-

mate-4-hydroxylase (C4H), 4-coumarate CoA ligase (4CL)

and chalcone isomerase (CHI) (Du et al., 2010; Chu et al.,

2014). Among these enzymes, IFS is directly involved in

the conversion of flavanone substrates to isoflavonoids

(Cheng et al., 2008). Interestingly, our transcriptome assay

of four tissues/organs in Y/Y and Y/y plants showed up-

regulation of the transcript abundance of nearly all of

the enzymes involved in isoflavonoid biosynthesis in the

12–14 mm seed and 14th trifoliolate leaf of Y/y plants com-pared with Y/Y plants (Figure 7a). Conversely, considerable

changes in transcripts of these enzymes were not observed

in the 8–10 mm seed of Y/y plants vs. Y/Y plants (Figure7a).

The increased transcripts of isoflavonoid biosynthesis

enzymes in the 12–14 mm seed of Y/y plants may improveseed accumulation of isoflavonoid products. Thus, we sub-

sequently assayed primary and secondary metabolites in

mature seeds harvested from Y/Y and Y/y plants (Tables

S4, S5). At the same time, primary and secondary metabo-

lites in the sprouts generated from these seeds were also

examined (Tables S6, S7) because sprouts are a common

and favorite food in Asia. Among the considerable changes

in a wide range of metabolites in the seeds and sprouts

between Y/Y and Y/y plants (Tables S4, S5, S6 and S7), the

mG

mC

HLI1

mG

mC

HLI2

AtCHLI1

GmCHLI1

GmCHLI2

GmCHLI3

mGmCHLI1

GmCHLI4

mGmCHLI2

BD

AtC

HLI1

Gm

CH

LI1

Gm

CH

LI2

Gm

CH

LI3

Gm

CH

LI4

AD

Prey

Bait

-Leu/-Trp/-His/-Ade

0

0.4

0.8

1.2

1.6

2

AD-GmCHLI1

β-ga

l act

ivity

(uni

t)

AD-mGmCHLI2AD-GmCHLI2

AD

AD-mGmCHLI1

bcab

d

a

e

bc

a

c

b

d

(a) (b)Figure 5. Interaction analysis among GmCHLIsusing the yeast two-hybrid system.

(a) Interactions among GmCHLIs. AtCHLI1 was used

as a positive control.

(b) Comparison of the interaction capacities of the

indicated proteins in yeast cells based on b-galassays. The error bar indicates the standard devia-

tion of three replicates. Statistical significance at

the 5% level was determined using Duncan’s multi-

ple range test.

© 2016 The AuthorsThe Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), doi: 10.1111/tpj.13282

Evolution of GmCHLIs in soybean 7

total content of isoflavonoids was found to be significantly

elevated in the seeds from Y/y plants (Figure 7b). However,

the genotype of the individual seed from Y/y plants had lit-

tle effect on the increase in isoflavonoids (Figure 7b). Simi-

lar results were observed for the sprouts (Figure 7b).

These findings suggest that the greater accumulation of

isoflavonoids in seeds from Y/y plants is mainly deter-

mined by maternal nutrition.

DISCUSSION

Evolution of oligomer-encoding GmCHLIs following

polyploidy in soybean

Polyploidy is one of the major forces promoting genome

evolution (Adams and Wendel, 2005; Flagel and Wendel,

2009), and clarification of how duplicated genes diverge

after polyploid events is important for understanding trait

determination. Mg-chelatase catalyzes the first critical step

in chlorophyll biosynthesis at the chlorophyll and heme

branch point of tetrapyrrole biosynthesis. The enzyme con-

sists of three distinct subunits: CHLI, CHLD and CHLH (Riss-

ler et al., 2002). The sequences and functions of these

subunits are highly conserved in different photosynthetic

organisms, and all three subunits are essential for proper

Mg-chelatase function (Hansson et al., 1999, 2002; Sawers

et al., 2006). It has been reported that CHLI needs to form

as a hexamer and then to interact with CHLD and CHLH to

function as a Mg-chelatase (Hansson et al., 2002; Masuda,

2008). Due to the two WGD events (Schmutz et al., 2010),

four CHLI paralogs, two CHLD paralogs and three CHLH

paralogs are present in the soybean genome; with the

exception of GmCHLH2, these genes are present as dupli-

cated pairs (Figure S2). Therefore, it would be useful to

determine how the different GmCHLI copies coordinate to

form a hexamer prior to becoming functional.

Our transgenic Arabidopsis assay demonstrated that all

four of the GmCHLIs can function as a CHLI subunit (Fig-

ure 4). However, our transcriptional investigation indicated

that the four copies have diverged into two pairs according

to expression patterns (Figure 2). Among these GmCHLIs,

GmCHLI1 and GmCHLI2 exhibited constantly high expres-

sion in most tissues, suggesting that they may be the

major CHLI isoforms. In contrast, GmCHLI3 and GmCHLI4

showed significantly low expression in most tissues, indi-

cating that they may no longer be the main genes encod-

ing the CHLI subunit in soybean, at least not in all tissues.

GmCHLI1 GmCHLI2 GmCHLI3

GmCHLI4 GmCHLD1 GmCHLD2

GmCHLH1 GmCHLH2 GmCHLH3

Y/Y Y/y y/y

00.010.020.030.040.050.06

0

0.02

0.04

0.06

0.08

0.10

0

0.0005

0.0010

0.0015

0.0020

0.0025

00.0020.0040.0060.0080.0100.012

0

0.05

0.10

0.15

0.200.25

00.0030.0060.0090.0120.0150.018

0

Root

Stem

Cotyl

edon

Unifo

liolat

e

leaf

Root

Stem

Cotyl

edon

Unifo

liolat

e

leaf

Root

Stem

Cotyl

edon

Unifo

liolat

e

leaf

369

121518

00.0010.0020.0030.0040.0050.0060.007

0

2

4

6

8

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Rel

ativ

e ex

pres

sion

Rel

ativ

e ex

pres

sion

Rel

ativ

e ex

pres

sion

Figure 6. Relative expression of GmCHLIs, GmCHLDs and GmCHLHs in the y mutant.

Expression patterns of GmCHLI1 (a), GmCHLI2 (b), GmCHLI3 (c), GmCHLI4 (d), GmCHLD1 (e), GmCHLD2 (f), GmCHLH1 (g), GmCHLH2 (h) and GmCHLH3 (i) in

different tissues/organs of the y mutant. RNA was isolated from seedlings when their unifoliolate leaves were fully expanded. ACT11 was used as an internal

control. The error bar indicates the standard deviation of at least three replicates.

© 2016 The AuthorsThe Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), doi: 10.1111/tpj.13282

8 Qing Li et al.

The similarity of GmCHLI1 and GmCHLI2 at both the mRNA

and protein levels suggest that these genes are functionally

redundant. Nevertheless, unlike other duplicated gene pairs

for which only a double mutant can generate a mutant phe-

notype (Fang et al., 2014), mutation in each copy of the

duplicated GmCHLI1 and GmCHLI2 pair can result in a

chlorophyll-deficient phenotype (Figures 1 and 3). This situ-

ation may be because all the GmCHLIs have the ability to

form both homo-hexamers and hetero-hexamers (Figures 5

and S5), and then further interact with GmCHLD and

GmCHLH to generate the Mg-chelatase complex.

Photosynthesis provides the energy required for the life

activities of almost all organisms and thus is one of the

most important biochemical reactions on Earth. Genetically

modifying genes involved in photosynthesis has long been

considered as a potentially efficient approach to increasing

crop yields, though it has met with significant challenges

(Horton, 2000; Long et al., 2006). Many proteins involved in

photosynthesis exist as oligomers to form functional com-

plexes (Liu et al., 2004; Guskov et al., 2009; Qin et al.,

2015; Suga et al., 2015), and even the chaperonins that

assist in protein assembly also need to function in a multi-

meric manner (Bai et al., 2015). It has been reported that

almost all crops have experienced polyploidy events (Wen-

del, 2000; Kondrashov et al., 2002), resulting in oligomer

formation by different duplicated copies. Interactions

among the subunits in a protein complex are critical for

activity, which could be affected by mutation in any of the

subunits (Zhong et al., 2003; Imai et al., 2008; Daras et al.,

2009; Kim et al., 2013; Kakehi et al., 2015; Zhang et al.,

2016), and our results support this notion. We found that

non-functional mGmCHLI1 or mGmCHLI2 strongly com-

peted with functional GmCHLIs in assembly of the CHLI

hexamer, thus forming a dysfunctional Mg-chelatase com-

plex (Figure 5). Therefore, we infer that the evolution of

duplicated oligomer-encoding genes will be more restricted

because they may be affected by other paralogs. This is

partly supported by the fact that in 302 resequenced soy-

bean accessions, no homozygous amino acid mutation is

found in conservative positions of the four GmCHLIs (Zhou

et al., 2015). Our results indicate that increasing crop yield

via modification of photosynthesis-related genes might be

a complex process, particularly for genes involved in multi-

mer formation because their modifications will affect coop-

erative interactions among different duplicated copies.

However, simultaneous modification of paralogs at the

same amino acid position is a potential strategy.

Application of GmCHLI1 for breeding

In addition to photosynthesis, the chloroplast is the site of

the biosynthesis of many metabolites such as organic

acids, amino acids and sugars. In general, abnormal

chloroplast ultrastructure would affect some metabolism

processes, thereby possibly altering the abundance of

metabolites in the leaves and other tissues/organs (Luo

et al., 2013; Satou et al., 2014). In this study, we found

impaired chloroplast ultrastructure in the chlorophyll-

deficient y mutant (Figure 1c), and our metabolomic data

revealed large changes in a number of metabolites in the

seeds and sprouts from heterozygosis mutant plants

(Tables S4, S5, S6 and S7). Interestingly, the total content

of isoflavonoids was significantly elevated in the seeds

and sprouts from Y/y plants (Figure 7b). As a class of

plant-specialized secondary metabolites that are almost

exclusive to the legume family, isoflavonoids play impor-

tant roles in plant–environment interactions and in humanhealth (Du et al., 2010). The increase of isoflavonoid levels

has long been considered in soybean breeding because of

their health-related benefits (Dhaubhadel et al., 2003). Our

investigation further revealed that the accumulation of iso-

flavonoids in the seeds and sprouts from Y/y plants is

mainly determined by the maternal genotype (Figure 7b).

(a)

(b)Seeds Sprouts

Y/y plants

0

10

20

30

40

50

0102030405060

Isof

lavo

noid

s (μ

mol

g–1

)

Isof

lavo

noid

s (μ

mol

g–1

)

eb d c a d

ab c b a

Y/Y plants Y/y plantsPALC4H4CL

CHSCHRCHIIFS

Y/y plants

Figure 7. Increased isoflavonoids in Y/y plants.

(a) Gene expression of the enzymes involved in isoflavonoid biosynthesis in

trifoliolate leaf and seed from Y/y and Y/Y plants. The total FPKM values of

the paralogs that encode the same enzyme were recognized as the mRNA

level for this enzyme, the data are shown in base 2 logarithmic form (log2Total FPKM).

(b) Total isoflavonoids in the mature seeds and sprouts from Y/y and Y/Y

plants. Total isoflavonoids are the sum of the mole content of each isoflavo-

noid type. Single Y/Y indicates seeds or sprouts harvested from Y/Y plants;

‘Y/y mix’ denotes seeds or sprouts of unknown genotype harvested from Y/

y plants; Y/Y, Y/y and y/y above the Y/y plants represent seeds or sprouts of

corresponding genotypes harvested from Y/y plants. The error bar indicates

the standard deviation of three replicates. Statistical significance at the 5%

level was determined using Duncan’s multiple range test.

© 2016 The AuthorsThe Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), doi: 10.1111/tpj.13282

Evolution of GmCHLIs in soybean 9

Similarly, a maternal effect on seed isoflavonoid content

was noted in the reciprocal crosses between soybean culti-

vars that differ in seed isoflavonoids (Dhaubhadel et al.,

2003). The isoflavonoid content of F1 seeds more closely

resembles that of the maternal parent. Moreover, the Y/y

heterozygosis mutant did not show a significant difference

in yield compared with wild-type plants. Therefore, Y/y

plants may be useful for future breeding of high-isoflavo-

noid soybean.

EXPERIMENTAL PROCEDURES

Plant materials and growth conditions

The soybean plants used for quantitative analysis of pigments,observation of chloroplast ultrastructure, determination of chloro-phyll fluorescence parameters and RT-qPCR were grown in agreenhouse (22–25°C, 16 h light/8 h dark photoperiod,130–180 lmol m�2 sec�1 light intensity and 50–60% relativehumidity). Soybean plants for map-based cloning, transcriptomeand metabolome analyses were grown at the farm of the Instituteof Genetics and Developmental Biology with the same cultivationmanagement. All of the Arabidopsis and N. benthamiana plantswere grown in a greenhouse (22–25°C, 16 h light/8 h dark pho-toperiod, 130–180 lmol m�2 sec�1 light intensity and 50–60% rela-tive humidity).

Pigments analysis

Chlorophylls and carotenoids were extracted using 100% acetoneand quantified spectrophotometrically using the following previ-ously published formulas (Lichtenthaler, 1987):

Concentration of chlorophyllaðCaÞ ¼ ð11:24A662� 2:04A645Þ � v/wConcentration of chlorophyllbðCbÞ ¼ ð20:13A645� 4:19A662Þ � v/w

Concentration ofcarotenoidsðCcarÞ ¼ðð1000A470� 1:90Ca� 63:14CbÞ=214Þ � v/w

(V indicates the volume of acetone; W represents the freshweight of the sample).

Transmission electron microscopy analysis

Leaf sections (approximately 1 mm 9 1 mm) from the same posi-tion were first placed in 1.5 mL primary fixation solution (2.5% glu-taraldehyde allocated with 0.1 M phosphate buffer) and maintainedunder vacuum for 3 h for full pre-fixation. After washing five timeswith 1 mL of 0.1 M phosphate buffer, the samples were post-fixedfor 2 h with 0.5 mL of 1% osmium tetroxide in the same buffer. Thesamples were then dehydrated in a graded acetone series and sub-sequently infiltrated with a graded epoxy resin series. The polymer-ization reaction was performed in 100% epoxy resin for 24 h at60°C. Ultra-thin sections were obtained using a Leica EM UC7 ultra-microtome (Leica, Wetzlar, Germany) and stained with uranyl acet-ate for 20 min and lead citrate for 5 min. The observations andrecording of images were performed using a JEM-1400 transmis-sion electron microscope (JEOL, Tokyo, Japan).

Chlorophyll fluorescence analysis

Fv/Fm, NPQ and qP were measured using an IMAGING-PAM M-series Chlorophyll Fluorometer with the MAXI version (HeinzWalz, Effeltrich, Germany). Plants were dark adapted for 30 min

before measurements. The operation and analysis were per-formed according to the manufacturer’s instructions.

Map-based cloning of the y locus

Heterozygous Y/y plants were crossed with the sequenced cultivarWilliams 82, and the pale-green F1 plants were selfed to generatethe F2 mapping population. DNA was isolated from F2 individualsusing the CTAB method. Positional cloning was performed usingthe combinations of sequence length polymorphism and cleavedamplified polymorphic sequence markers provided in Table S8.The markers were designed based on the DNA sequences of Wil-liams 82 and y mutant resequencing.

RNA extraction and RT-qPCR

Total RNA was extracted using an RNAprep Pure Plant Kit (TIAN-GEN, Beijing, China) according to the manufacturer’s instructions.cDNA synthesis was performed with M-MLV reverse transcriptase(Invitrogen, Carlsbad, CA, USA) in accordance with the manufac-turer’s protocol. RT-qPCR was performed using the LightCycler480system (Roche, Mannheim, Germany) and LightCycler� 480 SYBRGreen I Master Kit (Roche) according to the manufacturer’sinstructions. The relative expression level was calculated usingthe 2�DCt method. All of the primers used for RT-qPCR are listed inTable S9.

Plasmid construction

The plasmids used for different purposes, the construct methodsand their correspondingly amplified primers are listed inTable S10. For Arabidopsis transformation, the full-length CDSs ofAtCHLI1 and GmCHLIs were amplified using specific primers, dou-ble digested with XhoI and SpeI, and ligated into the correspond-ingly digested PFGC5941 vector. Similarly, the full-length CDSs ofAtCHLI1, GmCHLIs, GmCHLDs and GmCHLHs were cloned into thePucGFP vector without stop codons for fusion with GFP for subcel-lular location and into the pGADT7 and pGBKT7 vectors for theY2H assay by digestion and ligation using the combinations ofrestriction enzymes shown in Table S10. For the BIFC assay, thefull-length CDSs of AtCHLI1, GmCHLIs, GmCHLDs and GmCHLHswithout stop codons were first introduced into the entry vectorpCRTM8/GW/TOPO� by TA cloning and then transferred to the desti-nation vectors of nEYFP/pUGW2 and cEYFP/pUGW2 by recombina-tion using LR Clonase Enzyme (Invitrogen). For the promoteractivity analysis of AtCHLIs and GmCHLIs, the inferred promoters(from ~ 3 kb upstream of the 50 UTR to ATG start codon) werecloned into the pGreen-0800-LUC vector by digestion and ligationusing the combinations of restriction enzymes shown in Table S10.

Yeast two-hybrid assay

The Y2H assay was performed using the Matchmaker GAL4 Two-Hybrid System 3 (Clontech, Palo Alto, CA, USA) according to theClontech yeast protocols handbook. The full-length GmCHLIs werefused with both the GAL4 activation domain (AD) in pGADT7 andthe GAL4 binding domain (BD) in pGBKT7. Subsequently, differentcombinations of plasmids were co-transformed into yeast strainAH109. The resulting yeast co-transformants were first screenedat 30°C on plates containing double-dropout medium (-Leu/-Trp)and then scraped onto high-stringency plates containing quadru-ple-dropout medium (-Leu/-Trp/-Ade/-His) and grown at 30°C. Theb-gal assay for quantifying protein–protein interactions was per-formed according to the manufacturer’s instructions, with slightmodifications; chlorophenol red-b-D-galactopyranoside (CPRG,Roche) was used as the substrate.

© 2016 The AuthorsThe Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), doi: 10.1111/tpj.13282

10 Qing Li et al.

Subcellular localization and BIFC assay

To examine subcellular localization, PucGFP or its recombinantplasmid ligated to the target gene was transformed into Arabidop-sis protoplast cells as previously described (Yoo et al., 2007), withslight modifications. After incubation in the dark at 22°C for atleast 12 h, GFP fluorescence was observed using a confocal laserscanning microscope Leica SP8 (Leica). For the BIFC assay, Xgene-nEYFP/pUGW2 and Y gene-cEYFP/pUGW2 were co-trans-formed into Arabidopsis protoplast cells following the methoddescribed above, and YFP fluorescence was observed.

Transient transcription dual-luciferase assay

A transient Dual-Luciferase assay using N. benthamiana was per-formed following a previously described method (Hellens et al.,2005), with certain modifications. Agrobacterium EHA105 contain-ing a pGreen or pGreen-PCHLIs-LUC reporter plasmid was trans-formed into N. benthamiana leaves. After 3 days of incubation, asmall amount of the infected leaves were sprayed with 1 mMluciferin (Promega, Madison, WI, USA) supplemented with 0.01%Triton X-100 and photographed using a low-light cooled CCDimaging apparatus NightOWL II LB983 with Indigo software(BERTHOLD TECHNOLOGIES, Bad Wildbad, Germany) to assessLUC expression. The remaining leaves infected with the sametype of reporter plasmid were harvested and temporarily storedin liquid nitrogen for assay using the Dual-Luciferase� Reporter(DLRTM) Assay System (Promega). In the Dual-Luciferase assay,each sample was first ground into a powder using liquid nitro-gen, and ~ 50 mg of the powder was suspended and homoge-nized in 500 lL passive lysis buffer. A 20 lL aliquot of the crudeextract was mixed with 100 lL of LUC assay buffer, and LUCactivity was measured using a GloMax�-96 microplate lumin-ometer (Promega). Then, 100 lL of Stop and Glow buffer wasadded to test REN activity. Promoter activity was calculated bythe ratio of LUC to REN activities.

RNA sequence and data analysis

Sequencing libraries were constructed as previously described(Severin et al., 2010) and sequenced using an HiSeq 2500 system(Illumina, San Diego, CA, USA). The reads were mapped to thesoybean reference genome (Wm82. a2.v1) using HISAT (2.0.0)(Kim et al., 2015). Reference genome-referred transcript assemblyand calculation of the estimated expression was achieved with theStringTie (v1.0.4) package (Pertea et al., 2015). The FPKM valuewas used to calculate transcript abundance. As the enzymesinvolved in isoflavonoid biosynthesis in soybean are multi-copyencoded, to simplify the complexity of the transcript analysis ofthese enzymes, the total FPKM values of the multiple copies wererecognized as the mRNA level for each enzyme. The correspond-ing paralogs encoding the enzymes involved in isoflavonoidbiosynthesis and their FPKM values are shown in Table S11.

Metabolome analysis

Primary metabolites were measured using gas chromatography–mass spectrometry following a previously described method(Fiehn et al., 2000), with certain modifications. The relative massof each primary metabolite was calculated using ribitol as aninternal standard, and the unit of the relative mass is presented asmicrogram per gram of dry weight (lg g�1 dry weight). Secondarymetabolites were assessed using liquid chromatography–tandemmass spectrometry as previously described (Chang et al., 2012),with certain modifications. The relative mole content of each

secondary metabolite was calculated using daidzin as an externalstandard, and the unit of the relative mole content is presented asmicromole per gram of dry weight (lmol/g dry weight).

ACKNOWLEDGEMENTS

We thank Dr Li Hsou-min (Institute of Molecular Biology, Acade-mia Sinica) for kindly providing Arabidopsis cs215 seeds. Thiswork was supported by the National Key Research and Develop-ment Program for ‘Seven Crop Breeding’ Pilot Project (Grant No.2016YFD101001), the National Natural Science Foundation ofChina (Grant Nos. 91531304 and 31525018) and the ‘Strategic Pri-ority Research Program’ of the Chinese Academy of Sciences(Grant No. XDA08020202).

CONFLICT OF INTEREST

The authors have declared that no competing interests

exist.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article.Figure S1. Alignment of GmCHLI1 and related proteins.Figure S2. Duplications of GmCHLIs, GmCHLDs and GmCHLHs insoybean.Figure S3. Subcellular localization of GmCHLIs, GmCHLDs andGmCHLHs.Figure S4. Association analysis between the level of GmCHLIstransgenic expression and the chlorophyll content of transgenicplants.Figure S5. Interaction analysis among GmCHLIs by BIFC.Figure S6. Interaction analysis between GmCHLIs and GmCHLDsor GmCHLHs by BIFC.Figure S7. Interaction analysis among GmCHLDs, GmCHLHs, andGmCHLDs with GmCHLHs by BIFC.Figure S8. Relative expression of GmCHLIs, GmCHLDs andGmCHLHs in the y9 mutant.

Table S1. The gene information in map-based cloning locus.Table S2. The protein identity analysis of GmCHLIs, GmCHLDsand GmCHLHs.Table S3. The phenotype summary of the T1 transgenic plants.Table S4. The primary metabolome of y mutant seeds.Table S5. The secondary metabolome of y mutant seeds.Table S6. The primary metabolome of y mutant sprouts.Table S7. The secondary metabolome of y mutant sprouts.Table S8. Information of the molecular markers used for map-based cloning of the y locus.Table S9. Information of the RT-qPCR primers used in this study.Table S10. Information of the vectors used in this study.Table S11. Gene expression information for the enzymes involvedin the isoflavonoid biosynthesis pathway in the y mutant.

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