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ARTICLE
Received 8 Mar 2015 |Accepted 11 Dec 2015 |Published 22 Jan 2016
Genetic basis for glandular trichome formation in
cotton
Dan Ma1,*, Yan Hu1,*, Changqing Yang2,*, Bingliang Liu1, Lei Fang1, Qun Wan1, Wenhua Liang1, Gaofu Mei1,
Lingjian Wang2, Haiping Wang1, Linyun Ding1, Chenguang Dong1, Mengqiao Pan1, Jiedan Chen1, Sen Wang1,
Shuqi Chen1, Caiping Cai1, Xiefei Zhu1, Xueying Guan1, Baoliang Zhou1, Shuijin Zhu3, Jiawei Wang2,
Wangzhen Guo1, Xiaoya Chen2& Tianzhen Zhang1
Trichomes originate from epidermal cells and can be classified as either glandular or
non-glandular. Gossypium species are characterized by the presence of small and darkly
pigmented lysigenous glands that contain large amounts of gossypol. Here, using a dominant
glandless mutant, we characterize GoPGF, which encodes a basic helix-loop-helix
domain-containing transcription factor, that we propose is a positive regulator of gland
formation. Silencing GoPGF leads to a completely glandless phenotype. A single nucleotide
insertion in GoPGF, introducing a premature stop codon is found in the duplicate recessive
glandless mutant (gl
2
gl
3
). The characterization of GoPGF helps to unravel the regulatory
network of glandular structure biogenesis, and has implications for understanding the
production of secondary metabolites in glands. It also provides a potential molecular basis to
generate glandless seed and glanded cotton to not only supply fibre and oil but also provide a
source of protein for human consumption.
DOI: 10.1038/ncomms10456 OPEN
1State Key Laboratory of Crop Genetics and Germplasm Enhancement, Cotton Hybrid R & D Engineering Center (the Ministry of Education), Nanjing
Agricultural University, Nanjing 210095, China. 2National Key Laboratory of Plant Molecular Genetics, National Plant Gene Research Center, Institute of Plant
Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. 3Department of Agronomy,
College of Agriculture and Biotechnology, Zhejiang University, Zhejiang 310029, China. * These authors contributed equally to this work. Correspondence and
requests for materials should be addressed to T.Z. (email: cotton@njau.edu.cn) or to X.C. (email: xychen@sibs.ac.cn).
NATURE COMMUNICATIONS | 7:10456 | DOI: 10.1038/ncomms10456 | www.nature.com/naturecommunications 1
Trichomes are specialized structures that originate from
epidermal cells, and can be classified into two main
categories: non-glandular or glandular1. The most
remarkable feature of glandular trichomes is their unique
capacity to synthesize, store and sometimes secrete a wide array
of metabolites, including polysaccharides, organic acids, proteins,
terpenoids (such as the antimalarial drug artemisinin), alkaloids
and polyphenols1,2. These compounds give plants a distinctive
smell, the natural barrier to protect plants against herbivorous
insects and pathogens, or have a significant commercial value as
drugs, fragrances, food additives and insecticides1,3. As such,
glandular trichomes have been described as bio-factories for the
production of high-value natural products such as volatile oils,
resins, mucilages and gums. Despite extensive study of the
development and fine structure, and physiological and molecular
metabolisms with regard to chemicals synthesized, stored and
secreted in glandular trichomes4–7, almost nothing is known
about the genetics underlying their development.
Cotton (Gossypium. spp) is not only the leading natural fibre
resource, but also the third largest field crop in terms of edible
oilseed tonnage. In addition to 21% oil, cottonseed has a relatively
high protein content (23%). For every kilogram of fibre produced,
1.65 kg of seed is also collected. According to this ratio, the global
cottonseed production could potentially provide protein for half a
billion people annually8. However, the Gossypium species are
characterized by the presence of small darkly pigmented
lysigenous glands containing gossypol deposits, which are toxic
to humans and monogastric animals. Therefore, cottonseed
cannot be used to produce edible proteins or oils directly.
Gossypol is a yellowish phenolic compound that contributes to
the defence of cotton against pests, diseases and abiotic
stresses9,10. Therefore, potentially the best approach would be
to develop cotton that produces gossypol-free seed for human
consumption and normal gossypol content in other tissues for
protection from pests and pathogens by seed-specific genetic
engineering of gland formation or gossypol biosynthesis.
Ultra-low gossypol cottonseed lines have been developed using
RNAi knockdown of d-cadinene synthase gene(s) during seed
development in Gossypium hirsutum8. The ultra-low gossypol
trait is stable under field conditions and the foliage/floral organs
contain wild-type levels of gossypol and related terpenoids,
indicates it could be possible to enhance and expand the
nutritional utility of the annual cottonseed output to fulfil the
ever-increasing needs of humanity after the evaluation of its
safety and nutritional efficacy9.
Understanding the molecular genetic basis of gossypol gland
formation could provide additional methods to develop
gossypol-free cotton seeds that could be produced efficiently
and be adapted widely. The glands originate from a cluster of
gland primodium cells, which differ from other cells in that they
have a high-density cytoplasm and large nucleolus. The internal
cells are then degraded and form a cavity known as a gland in the
ground meristem11. From a cross between a cultivated and a
primitive race of G. hirsutum, McMichael12,13 recovered a
recessive mutant that eliminates all glands on the aerial plant
parts and seeds (Fig. 1). This offered the possibility of cultivating
glandless cotton, which could potentially help to alleviate hunger
and protein shortages14. Up to now, genetic research has revealed
that cotton gland formation is determined by a combination of at
least six independent loci, gl
1
,gl
2
,gl
3
,gl
4
,gl
5
and gl
6
(ref. 15). The
glandless phenotype is controlled by two pairs of duplicate
recessive genes (gl
2
gl
3
) on chromosome (chr.) A12 and D12,
respectively15,16. In addition, a single dominant glandless
mutation in G. barbadense was discovered in Egypt following
the irradiation of Giza 45 seeds with 32P, which results in
glandless plants and seeds (Figs 1 and 2a). Genetic analysis
revealed that it is a dominant allele at the Gl
2
locus that is
epistatic to Gl
3
, and the gene symbol Gl
2
ewas proposed17,18.
A new strain of cotton that is homozygous for this new gene was
released as Bahtim 110 in Egypt19 and Hai-1 in China18. These
two mutants have been used to develop many glandless cultivars
of both G. hirsutum and G. barbadense to produce little or no
gossypol in seeds. For example, more than 20 glandless cultivars
have been developed in China20. However, the molecular genetic
basis for the formation of pigment glandular trichomes, which are
storage organs of gossypol in cotton, remains unknown. Here,
through a map-based cloning approach, we identify Gossypium
PIGMENT GLAND FORMATION GENE (GoPGF) as the likely
causative gene for the phenotype of glandless mutant cotton. This
helps to uncover the molecular basis for formation of glandular
trichomes and secondary substances such as gossypol in cotton.
Results
Map-based cloning of the dominant glandless gene Gl
2
e.We
previously anchored the dominant glandless gene Gl
2
ebetween two
microsatellites (SSRs), NAU3778 and NAU2251b, with genetic
distances of 9.27 and 0.96 cm, respectively in chr. 12A (ref. 21). On
thebasisofthegenomesequenceofG. raimondii22,Gl
2
ewas
delimited within a 1-Mb interval on scaffold D8. We mined and
developed 316 SSRs to screen the polymorphism between Hai-1 and
TM-1 within this region (Supplementary Data 1). We used an
enlarged mapping population including 2,197 individuals
(Supplementary Table 1) and seven polymorphic SSRs, narrowed
the Gl
2
elocus to a 43-kb region flanked between w7954 and w5383
with genetic distances of 0.5 and 0.6 cM, respectively (Fig. 2b;
Supplementary Data 1). Within this region, seven putative open
reading frames (ORFs; Supplementary Table 2) are predicted based
on our high-quality reference genome sequence of tetraploid cotton
G. hirsutum acc. TM-1 (ref. 23). Quantitative RT–PCR (qPCR;
Fig. 2c; Supplementary Fig. 1) revealed that only the expression level
of ORF2 was altered, and was significantly lower in the dominant
glandless mutant Hai-1 and the (Hai-1 TM-1)F
1
than in the
glanded G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124.
Therefore, we considered ORF2 as a candidate gene for Gl
2
e.
We then isolated the full length genomic DNA of ORF2 from the
glandless Hai-1 and its wild type, Giza 45, and found that the
coding region is 1,428 bp in length with no intron and contains a
predicted gene of unknown function. The protein is predicted to
contain a conserved motif shared by bHLH and R2R3-MYB in the
N-terminal motif (Pfam14215) from amino acid 20 to 202 and a
helix-loop-helix DNA-binding domain (Pfam00010) from amino
acid 293 to 350 at the C-terminal (http://pfam.sanger.ac.uk/), which
is involved in DNA binding and protein oligomerization. The gene
is phylogenetically closely related to bHLH transcription factor
(Thecc1EG015640t1) in Theobroma cacao and belongs to bHLH
transcription factor family. We named the gene as Gossypium
pigment gland formation gene (GoPGF). Orthologous genes of
GoPGF in G. raimondii and G. arboreum are Gorai.008G259000.1
and Cotton_A_01306, respectively. GoPGF protein was localized to
the nucleus (Supplementary Fig. 2). Quantitative reverse transcrip-
tase PCR (qRT–PCR) analysis revealed that the gene is expressed in
a constitutive manner in most organs and tissues, including root,
stem and leaf (Fig. 2c; Supplementary Fig. 3), consistent with the
presence of glands. DNA sequence comparisons revealed that three
single nucleotide polymorphisms (SNPs) exist between the
dominant glandless Hai-1 and the glanded Giza 45 and other
G. barbadense cultivars in GbPGF (Supplementary Fig. 4), but only
result in one amino acid change from alanine to valine at residue 43
(Fig.2d).OnthebasisoftheseSNPs,wefoundthatthemutant
allele (GbPGFm) from Hai-1 co-segregates with 1,624 glandless
plants of three F
2
s(SupplementaryData1).
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10456
2NATURE COMMUNICATIONS | 7:10456 | DOI: 10.1038/ncomms10456 | www.nature.com/naturecommunications
The difference in GoPGF mRNA levels between Hai-1 and
TM-1 may possibly be determined by transcriptional regulation
of the GoPGF gene. To investigate the differential expression of
GoPGF_A12 from TM-1 and Hai-1, we isolated 1.8-kb fragments
upstream of the start codon of GoPGF from TM-1 and Hai-1. No
difference in transient expression of GUS reporter constructs was
detected after Agrobacterium-mediated transformation of tobacco
leaves (Supplementary Fig. 5), indicating that their promoters
likely do not confer the extra low expression of PGF in Hai-1.
Whether the mutation reduces its expression in Bahtim 110 or
Hai-1 or whether a causative mutation on a distant loci24 exerts
influence on GbPGF remains to be explored.
Nucleotide insertion results in two recessive mutant alleles. The
whole plant glandless phenotype is controlled by a combination
of two recessive mutant alleles, gl
2
and gl
3
(ref. 13) or one
dominant gene Gl
2
e(refs 13,15; Fig. 1). So, if PGF is the candidate
gene for Gl
2
e, we reasoned that PGF_A12 and PGF_D12 in the two
recessive mutant alleles, gl
2
and gl
3
, should be non-functional.
We isolated and sequenced GoPGFs from tetraploid cotton
accessions and their extant progenitor species, G. arboreum (A
2
)
and G. raimondii (D
5
). Sequence alignments show that there
are homoeologous PGF gene pairs in the corresponding
A subgenome (GoPGF_A12) and D subgenome (GoPGF_D12)of
tetraploid cottons with seven SNPs between them
(Supplementary Fig. 4; Supplementary Table 3). We isolated the
mutant GoPGF genes in the chr. A12 (GhPGF_A12m) and D12
(GhPGF_D12m), respectively, from the duplicate recessive
glandless mutant, 2(gl
2
gl
3
), and the monomeric mutants 2(Gl
2
gl
3
)
and 2(gl
2
Gl
3
) with a low number of glands (Fig. 1). Sequence
alignments show that a single ‘‘T’’ nucleotide insertion occurs
between 735 and 736 bp in the coding region of GhPGF_A12m
(gl
2
) in chr. A12, and a single ‘‘A’’ nucleotide insertion occurs
between 916 and 917 bp in the coding region of GhPGF_D12m
(gl
3
) in chr.D12. This premature translation termination corre-
lated with the production of fewer glands in monomeric mutants
(Gl
2
gl
3
and gl
2
Gl
3
) or completely glandless phenotype in the
duplicate mutant (gl
2
gl
3
; Figs 1 and 2d; Supplementary Fig. 4).
qPCR analysis revealed that GhPGF_A12m(gl
2
) and
GhPGF_D12m(gl
3
) were expressed at very low levels compared to
their corresponding homoeologous genes (Gl
3
and Gl
2
) in the
monomeric mutants 2(gl
2
Gl
3
) and 2(Gl
2
gl
3
), respectively, as well
as in the duplicate recessive glandless mutant 2(gl
2
gl
3
) and in
Hai-1 (Fig. 2e). These results strongly suggest that GoPGF is the
gene that controls cotton pigment gland formation.
GbPGF-silencing results in a glandless phenotype. To further
assess its function, we cloned the 30-end fragment (904–1,428 bp)
of GbPGF_A12 from Giza 45 and inserted it into pTRV2 for
virus-induced gene silencing (VIGS)25 to suppress the expression
Dominant
mutation
A12
Gl2e
Gl2e
Gl3
Gl3
D12
Dominant glandless cotton, 2(Gl2
eGl3)
A12 D12
Gl2
Gl2
Gl3
Gl3
Wild-type glanded cotton, 2(Gl2Gl3)
Recessive
mutation Recessive
mutation
A12
gl2
gl2
Gl3
Gl3
D12 A12 D12
Gl2
Gl2
gl3
gl3
Less glanded cotton, 2(gl2Gl3)Less glanded cotton, 2(Gl2gl3)
A12
gl2
gl2
gl3
gl3
D12
Duplicate recessive
glandless cotton, 2(gl2 gl3 )
X
Figure 1 | Origins of the genotypes and phenotypes of glandless tetraploid cottons. The genotypes include a dominant mutation, Gl
2
eGl
3
; two recessive
mutations, gl
2
Gl
3
and Gl
2
gl
3
; and a duplicated recessive cotton, gl
2
gl
3
. Black points in each graph show the glands on the surface of seeds.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10456 ARTICLE
NATURE COMMUNICATIONS | 7:10456 | DOI: 10.1038/ncomms10456 | www.nature.com/naturecommunications 3
of endogenous GbPGF in two cultivated glanded tetraploid
cottons. The PGF-silenced G. barbadense cv. Giza 45 (wild type of
the Hai-1 mutant), Hai7124 and G. hirsutum acc.TM-1 plants all
exhibited a glandless or low gland number phenotype in the
newly emerging tissues 14 days post-agro-infiltration (Figs 3a and
4; Supplementary Fig. 6). We observe no or fewer visible glands in
the new growing upper stems and leaves of the PGF-silenced
plants, but the stems below the cotyledon node and cotyledons
had many thickly dotted glands that had already formed before
infiltration. The transcripts of GbPGF in the PGF-silenced leaves
were significantly reduced compared to the untreated TM-1 and
Hai7124, indicating that GbPGF was effectively silenced in VIGS
plants (Figs 3b and 4a). In the wild-type cotton plants, large
cavities of mature pigmented glands were present, however, no
such gland cavities were observed in the mesophyll cells of
GbPGF-silenced leaves in either Hai7124 or TM-1 (Fig. 3c). These
data further suggest that GbPGF regulates the formation of glands
that act as a storage organ for gossypol and other related
sesquiterpenes in cotton. Consistently, by suppressing the GoPGF
expression, gland cell differentiation was blocked, and
hemigossypol and gosypol content was reduced by over 93% in
the GbPGF-silenced leaves compared with the untreated TM-1
and Hai7124 leaves (Fig. 3d), suggesting that PGF is also involved
in gossypol biosynthesis, directly or indirectly. Interestingly,
however, the non-glandular hair trichomes developed normally
(Supplementary Fig. 7) in the PGF-silenced G. hirsutum,
suggesting that development of hair or non-grandular and
grandular trichomes may use different genetic machinery in
cotton.
Gossypol biosynthesis is not linked to gland formation.
Through an antisense strategy, we developed transgenic
cotton plants with seed-specific silencing of ( þ)-d-cadinene-8-
hydroxylase (CYP706B1), a P450 monooxygenase in the gossypol
biosynthesis pathway26. Gossypol levels were significantly
reduced in the transgenic seeds, but lysigenous glands still
formed as in non-transgenic plants (Supplementary Fig. 8). This
result is similar to a previous report, which showed that silencing
of ( þ)-d-cadinene synthase gene (CDN)27 that catalyses the first
step in gossypol biosynthesis did not affect gland formation,
suggesting the terpenoid aldehyde synthesis and gland formation
are uncoupled.
Phylogenetic analysis shows a distinct ‘glandular trichome
formation’ clade. Together with other bHLH members in the
vascular plants species covered by glandular trichome such as
Nicotiana tabacum,Solanum lycopersicum and Artemisia annua
(Supplementary Fig. 9; Supplementary Data 2), GoPGF is
TM-1 Hai-1 12S 12L
NAU2251 W5383 W7954
W6423
W6421 W6451 W6439 W1073 CIR362
0.47 0.48 0.59 0.52 0.58 0.47 3.07 8.24 (cm)
43 kb
ORF1 ORF2 ORF3 ORF4 ORF5 ORF6 ORF7
N-terminal motif Basic helix loop helix
Val
Ala
Ala
Ala
Ala
475 aa
475 aa
245 aa
475 aa
309 aa
A12
gl2
Gl2
Gl2e
gl3
Gl3
D12
ORF 2
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Relative expression level
RSL RSL RS LRS L
H7124 TM-1 Hai-1 Hai-1 ×
TM-1
0.4
0.3
0.2
0.1
0
Expression level of GOPGF
RSL RS LRS LR S LR S L
TM-1 Hai 1 Gl2gl3 gl2Gl3 gl2gl3
GoPGF
A/D
GoPGF_A12 GoPGF_D12
Gl2e
Figure 2 | Fine mapping of dominant glandless gene Gl and the expression pattern of GoPGF.(a) Gland traits of seeds, leaves and stems of TM-1
(glanded) and Hai-1 (glandless). (b) Fine mapping of the dominant glandless gene, Gl. Seven genes (ORF1–7) in the mapping region are indicated by boxes.
(c) Real-time RT–PCR expression analysis of ORF2 in the root, stem and leaf of Hai7124, TM-1, Hai-1 and Hai-1 TM-1. Error bars represent the s.d. of the
mean values of three biological replicates. (d) Sequence diversity of PGF. An amino acid change from alanine to valine was observed in the dominant
glandless gene (Gl). The premature translation termination resulted in the production of fewer glands (Gl
2
gl
3
and gl
2
Gl
3
mutant) or completely glandless
(gl
2
gl
3
mutant) phenotypes. (e) Pyrosequencing analysis of the relative expression levels of GoPGF_A12 and GoPGF_D12 homoeologous alleles in the root,
stem and leaf of TM-1, Hai-1, 2(Gl
2
gl
3
), 2(gl
2
Gl
3
) and 2(gl
2
gl
3
).
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10456
4NATURE COMMUNICATIONS | 7:10456 | DOI: 10.1038/ncomms10456 | www.nature.com/naturecommunications
classified in a distinct clade (clade II), which has 19 unknown
function members. These bHLHs form a specific cluster involved
in regulating glandular trichome formation, distinct from other
bHLHs known to be involved in secondary metabolism such as
AtMYC2,AtMYC3 and AtMYC4 in Arabidopsis28 (Fig. 5a).
Differential gene expression in GoPGF-silenced plants. To gain
insight into the regulatory networks that may underlie gland
formation, we compared gene expression in leaves of
GoPGF-silenced plants, the control TM-1 and the mutant Hai-1
by RNA sequencing (RNA-seq). We found that 3,582 genes were
deferentially expressed with 2,276 upregulated and 1,306
downregulated in the GhPGF-silenced leaves, including
significantly reduced expression of 15 terpenoid synthase (TPS)
genes, 18 MYBs and 31 WRKYs (Supplementary Data 3). The
upregulated genes include genes related to light reaction, cell wall
degradation and protein synthesis. Strikingly, the downregulated
genes include genes related to secondary metabolism and terpe-
noid biosynthesis29, jasmonate (JA) signalling and genes in the
WRKY and MYB transcription factor families (Supplementary
Fig. 10). Subsequent qPCR confirmed decreased expression of
some TPS and WRKY genes in PGF-silenced tissues
(Supplementary Fig. 11). Yeast one-hybrid binding analysis
suggests that GoPGF could specifically interact with the G-box
motif (Supplementary Fig. 12), which is commonly found in TPSs
and WRKYs promoter regions including CDN-1 (ref. 29).
Pathway analysis also revealed these differentially expressed
genes included genes involved in secondary metabolism
(Supplementary Table 4). We also found that expression of
GoPGF was induced by JA treatment (Supplementary Fig. 13).
We speculate that GoPGF protein could control the specification
TRV:00 TRV:PGF
TRV:CLA1 TRV:00 TRV:PGF Hai -1
TRV:CLA1 TRV:00 TRV:PGF Hai-1
0.3
0.25
0.2
0.15
0.1
0.05
0CK TRV:PGF CK TRV:PGF
TM-1
H7124
** **
Relative expression level
of PGF
1.5
1.2
0.9
0.6
0.3
0CK TRV:00 #1 #2
** **
Content of hemigossypol (%)
0.2
0.15
0.1
0.05
0
CK TRV:00 #1 #2
** **
Content of gossypol (%)
Figure 3 | Functional characterization of GoPGF by VIGS. (a) Phenotypes of TM-1 before and after GoPGF silencing by VIGS, showing the presence
and the absence of the glands. (b) Transcript level of GoPGF in GoPGF-silenced leaves. (c) Cavity observed in the leaves of TM-1 but disappeared in the
leaves emerged after VIGS. Scale bar, 10 mm. (d) Hemigossypol and gossypol content in control (CK), empty vector (TRV:00) and in the GoPGF-silenced
(#1 and #2) leaves of TM-1, determined by high-performance liquid chromatography. **Po0.01; Student’s t-test, n¼3. Error bars are s.d. of three biological
repeats.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10456 ARTICLE
NATURE COMMUNICATIONS | 7:10456 | DOI: 10.1038/ncomms10456 | www.nature.com/naturecommunications 5
and differentiation of gland cells, possibly through regulating the
expression of JAZ,WRKYs or other genes. In addition, PGF may
also regulate sesquiterpene biosynthesis via binding to the
promoters of TPSs or WRKYs (Fig. 5b).
Discussion
We have provided evidence that GoPGF, encoding a bHLH
transcription factor, is likely the causative gene for the glandless
phenotype in cotton and appears to be a regulator of glandular
trichome formation. It therefore likely represents the first gene to
be successfully cloned in tetraploid cotton using a map-based
cloning strategy. Its cloning helps to elucidate the molecular
mechanism of the genetic control networks involved in secondary
substances and formation of glandular trichome, which
are storage organs in other plants such as N. tabacum30,
S. lycopersicum31,A. annua32 and so on. We identified a bHLH
cluster that may have diverged from other bHLH genes and be
involved in grandular trichome formation. Glandular trichomes
are metabolic hotspots for biosynthesis, regulation and release of
numerous volatile and non-volatile phytochemicals used by
plants for interacting with the biotic environment. Natural
products synthesized in trichomes are also widely adapted as
flavorants, perfumes and pharmaceuticals. The distinct glandular
trichome formation clade will provide us important candidates to
control the biosynthesis of useful secondary compounds through
genetic engineering.
Our results further clarified the close and complicated
relationship between gland and gossypol. Our study showed
glands developed normally in CYP706B1-silenced transgenic
cotton with reduced gossypol content, suggesting that gossypol
was not required for gland morphogenesis, indicating they were
uncoupled and controlled by different molecular machines.
Nevertheless, gossypol content was markedly suppressed in the
GoPGF-suppressed cotton without noticeable glands, suggesting
that suppression of gland formation will feedback to gossypol
0.0025
0.002
0.0015
0.001
0.0005
0
**
Giza 45 Giza 45-
VIGS
Relative expression level
Giza 45-
VIGS
Giza 45 Giza 45Giza 45-VIGS
Figure 4 | Functional characterization of GoPGF by VIGS in G. barbadense
cv. Giza 45. (a) Transcript level of GoPGF in normal Giza 45 and
corresponding GoPGF-silenced leaves. Error bars are s.d. of three
biological repeats. **Po0.01; Student’s t-test, n¼3. (b,c) Phenotypes
of Giza 45 before and after GoPGF silencing by VIGS. Yellow and blue
arrows indicate the cotyledonary node and the first vegetative branch,
respectively.
26
70
14
8
7GabHLH03
GhbHLH12/GoGPF
GrbHLH12
GhbHLH25
GhbHLH24
CcbHLH01
PtbHLH01
RcbHLH01
JcbHLH01
GabHLH10
GhbHLH03
GhbHLH15
GrbHLH03
TcbHLH03
HbbHLH01
SIbHLH08
CsbHLH01/MYC4-like
SIbHLH09
NtbHLH01
53
41
46
93
16
73
23
11 36
31
67
Signal
050
TPH
GoPFG FPP synthase
GaWRKY1
JAZs WRKYs
TPH TPH
Others
genes?
Gland
TPH
TPH
IPP
Acetyl-CoA
HMG-CoA
DMAPP TPH
HMGR
FPP
TPS
CYP706B1
(+)-δ-Cadinene
8-Hydroxy-(+)-cadinene
Hemigossypol
Gossypol
Figure 5 | A proposed model of gossypol biosynthesis and gland formation. (a) Among 111 bHLH members (Supplementary Data 2), 19 bHLHs from the
distinct clade II may be responsible for glandular trichome formation. Numbers on branch nodes indicate percentage support in 1,000 bootstrap trials.
(b) A proposed model of gossypol biosynthesis and gland formation. Blue text indicates genes downregulated in GoPGF-silenced TM-1. Heat map shows
gene expression level with FPKM value in VIGS-free TM-1 (AT), PGF-silenced TM-1 (P) and Hai-1 (H), each block indicates one gene. First, the expression of
GoPGF is induced by exogenous signal including JA. Then, GoPGF protein, as a regulator, controls the specification and differentiation of gland cells through
regulating the expression of JAZ,WRKYs or other genes. On the other hand, GoPGF can specifically interact with the G-box motif, which is commonly found
in TPSs and WRKYs promoter regions to feedback on gossypol biosynthesis pathway.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10456
6NATURE COMMUNICATIONS | 7:10456 | DOI: 10.1038/ncomms10456 | www.nature.com/naturecommunications
biosynthesis possibly through regulating the expression of
gossypol-related genes such as TPSs and WRKYs by binding to
their promoters. Taken together, cloning and functional assess-
ment of PGF will open novel opportunities to decipher the
mechanism of glandular trichome development in plants.
Australian Gossypium species such as G. australe,G. bickii and
G. sturtianum, contain immature lysigenous glands but no
terpenoid aldehydes, so the pigment glands only appear after seed
germination; thus, the dormant seeds of these species lack
gossypol33,34. This distinguishing characteristic, known as the
delayed gland morphogenesis trait, has the potential to enable the
large scale, direct usage of cottonseed. Various efforts have been
made to introduce this unique characteristic of wild Australian
cotton species into cultivated tetraploid cotton35, but such
cultivars with the delayed gland morphogenesis trait have not
been developed in cultivated tetraploid cottons by traditional
breeding methods. The cloning of GoPGF may offer the
opportunity to develop such cultivars as we investigate the role
of GoPGF in the delayed gland formation in Australian species.
These discoveries may help further to improve the productivity
and economic value of cotton.
Methods
Plant materials.A mutant, ‘‘Hai-1’’ (G. barbadense), with a glandless trait
controlled by a dominant gene, Gl
2
e(refs 17,19), was gifted by the Cotton Research
Institute, Chinese Academy of Agricultural Science (CAAS). N1 and N7 are near
isogenic lines of dominant glandless traits in Upland cotton. Three F
2
mapping
populations of 1,599, 244 and 354 individuals, respectively, were developed by
crossing TM-1 as a female parent to three glandless lines; Hai-1, N1 and N7. The
gland traits of leaves were investigated in 2,197 individuals. TM-1 (G. hirsutum),
Giza 45 and Hai7124 (G. barbadense), which have glands throughout the plants, and
three other Upland cotton germplasms, 2(Gl
2
gl
3
) and 2(gl
2
Gl
3
), which have a few
glands, and 2(gl
2
gl
3
), which has no glands, were used in this study. All cultivars were
planted at Jiangpu Experiment Station and in green houses at Nanjing Agriculture
University. Fresh leaves from the cultivars served as the source of genomic DNA, and
other vegetative and reproductive tissues were collected for total RNA extraction.
RNA was also extracted from developing embryos excised from ovules obtained
from each boll at 3, 0, 3, 5, 10 and 20 days postanthesis. All collected plant materials
were immediately frozen in liquid nitrogen and stored at 70 °C.
Map-based cloning of Gl
2
egene.A previous study reported that the Gl
2
elocus lies
between the molecular markers CLR362, NAU2251b, NAU3860b and STV033, with
genetic distances of 9.27 and 0.96 cm21, respectively. The genetic markers were plotted
to the scaffolds of G. raimondii22.TheGl
2
elocus was mapped to a 1-Mb region flanked
by CLR362 and NAU2251b on scaffold D8, namely chr. 12, based on the linkage map
constructed by JoinMap 3.0 (ref. 36). By using 598 F
2
mutant plants with additional
molecular markers developed in this work (Supplementary Data 1), the Gl
2
elocus was
further mapped to a 43-kb region between SSR marker w7954 and w5383. Then, the
genomic DNA sequence of the candidate gene was amplified from G. arboreum cv.
Jianglinzhongmian (A genome), G. raimondii (D genome), TM-1, Giza 36, Giza 45,
Giza 67, Giza 80, Junhai-1, Hai7124 and Hai-1 (A
t
D
t
genome) using primers
(Supplementary Data 1), and the PCR products were confirmed by sequencing.
Subcellular localization of GoPGF.To examine the subcellular localization of
GoPGF in cells, the PCR fragment amplified from cDNA from Hai-1 using the
primers K0016F and K0016R (Supplementary Data 1) and inserted into transient
expression vector pBinGFP4 (ref. 37) and generated the constructs
pBinGFP4::GoPGF. The constructs were introduced into Agrobacterium
tumefaciens strain GV3101 by electroporation. The recombinant plasmid was
introduced into tobacco (Nicotiana benthamiana) leaf cells by A. tumefaciens
infiltration37. Green fluorescent protein signals in the tobacco epidermal cell were
examined and photographed using a ZEISS LSM 710 confocal microscope (Zeiss
Microsystems) with a 20 objective lens (Zeiss) at the specific excitation and
emission waveleng ths 488 and 495–530 nm.
Quantitative RT–PCR analysis and pyrosequencing.RNA was extracted from
the different tissues (root, stem, leaf and embryos) from plants with and without
glands using a BioFlux kit. First-strand cDNA was generated using TransScript
One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotec Co.,
Ltd.) according to the manufacturer’s instructions. Quantitative RT–PCR and
pyrosequencing were performed with the primers listed in Supplementary
Data 1. The pyrosequencing reaction was performed using a PyroMark sequencer
(Qiagen, Valencia, CA).
Promoter activity analysis.The GoPGF promoter was isolated with the designed
primers H3996F and H3996R from the A subgenome of TM-1 and Hai-1
(Supplementary Data 1). The promoters cover 1,818 and 1,809 bp in length
upstream of the transcription start site. The binary vector pBI121 was used as a
basic expression vector to make constructs. All the constructs were introduced into
A. tumefaciens strain GV3101. Individual Agrobacterium colonies were grown on
Luria-Bertani (LB) plates with kanamycin (50 mgml1) for 48 h at 28 °C. A single
positive colony was used to inoculate a 5-ml culture (LB with 50 mgml1
kanamycin). Bacteria were pelleted, resuspended in infiltration medium (10 mM
MgCl
2
, 10 mM MES and 150 mM acetosyringone (pH 5.6)) to an OD
600
of 0.5–0.6,
then incubated at room temperature for 3 h. The bacterial suspension was infil-
trated into the abaxial side of fully expanded 6-week-old N. benthamiana leaves
using a needleless 1-ml syringe. For each experiment, the positive control (pBI121
intron with 35S-GUS), the negative control (TATA-GUS in pMDC162 intron), and
the constructs under investigation were infiltrated in areas of the same leaf or
different leaves. After infiltration, the plants were kept in the greenhouse for
72 h for inoculation. For histochemical staining, the plant tissues were incubated
at 37 °C overnight (12 h) in the dark in 1mM X-Gluc (5-bromo-4-chloro-3-
indolyl-b-D-glucuronide) in 100 mM sodium phosphate (pH 7.0), 10 mM EDTA,
0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.3% (v/v) Triton
X-100 and 20% (v/v) methanol to eliminate end ogenous GUS activity . After 12 h of
staining, tissues were destained in an ethanol series (50, 70 and 95%) to remove
chlorophyll, stored in 70% (v/v) ethanol, and photographed with a digital camera38.
Virus-induced gene silencing assay.To knockdown the expression of PGF gene,
a 415-bp fragment of GbPGF_A12 cDNA from TM-1 corresponding to bases
904–1,319 of the PGF gene was PCR-amplified using Pfu DNA polymerase
(Promega) and primers H1238F and H1238R (Supplementary Data 1). The
resulting PCR product was cloned into XbaI-BamH I-cut pTRV2 (ref. 25) to
produce a VIGS vector named pTRV2-PGF_A12. The vectors pTRV1 and pTRV2-
PGF_A12 were introduced into the Agrobacterium strain GV3101 by
electroporation (Bio-Rad, Hercules, CA, USA). For the VIGS assay, the
transformed Agrobacterium colonies containing pTRV1 and pTRV2-PGF_A12
were grown overnight at 28 °C in an antibiotic selection medium containing
rifampicin and kanamycin 50 mg l 1each. Agrobacterium cells were collected and
resuspended in infiltration medium (10 mM MgCl
2
, 10 mM MES and 200 mM
acetosyringone), adjusted to an OD
600
0.5. Agrobacterium strains containing TRV1
and TRV2 vectors were mixed at a ratio of 1:1. Seedlings with mature cotyledons
but without a visible rosette leaf (7 days after germination) were infiltrated by
inserting the Agrobacterium suspension into the cotyledons via a syringe. The
plants were grown in pots at 25 °C in a growth chamber under a 16-h light per 8-h
dark cycle with 60% humidity.
Histochemistry and microscopy.Leaves detached from the seedlings before and
after the treatment were cut into 1 mm2pieces and fixed in 0.1 mol l 1phosphate
buffer (pH 7.0) containing 2.5% glutaraldehyde at 4 °C overnight. After three
30-min rinses in 0.1 mol l 1phosphate buffer (pH 7.0), samples were postfixed in
0.5% osmium tetroxide solution in buffer at 4 °C for 3 h. The samples were then
rinsed in 0.1 mol l 1phosphate buffer (pH 7.0) for 30 min three times. Samples
were dehydrated through a series of ethanol solutions (30, 40, 50, 65, 80, 90 and
100%; 30 min in each) twice, with a final change to 1, 2-epoxypropane, and were
then embedded in Epon 812. Semi-thin sections (1–2 mm) were cut using a
Reichert-Jung ultramicrotome and stained with toluidine blue O or methylene blue.
Sections were examined and digitally recorded on a microscope.
Gossypol detection and analysis.The total gossypol concentration in leaves from
TM-1, PGF-silenced TM-1, Hai-1, 2(Gl
2
gl
3
), 2(gl
2
Gl
3
) and 2(gl
2
gl
3
) plants was
determined by high-performance liquid chromatography (HPLC)39. Each 100 mg
of freeze-dried plant sample was dissolved with 1 ml leaf extraction (acetonitrile/
water/phosphoric acid ¼80:20:0.1) for 1 h. The leaf extraction was centrifuged at
low speed for 5 min and then transferred the supernatant carefully at room
temperature. The eluent was filtered through a 0.22-mm nylon filter into a vial for
HPLC analysis with Agilent Zorbax eclipse XDB-C18 analytical column
(150 4.6 mm, 5 micron). The column was eluted with buf fer (EtOH/MeOH/IPA/
ACN/H
2
O/EtOAc/DMF/PPAcD ¼16.7:4.6:12.1:20.2:37.4:3.8:5.1:0.1) and kept at
35±1°C during the procedure. The determination wave length was 272 nm.
Standards of gossypol, hemigossypolone, were used to assess the retention time of
the individual terpenoids. The concentration of these compounds was calculated
using Agilent 1100 system by comparing to the gossypol standard curve. All the
reagents were of analytical grade and were made in China, with the exception of
gossypol, which was purchased from Sigma Chemical Co.
RNA-seq.Total RNA samples were quality-checked using RNA Pico Chips on an
Agilent 2100 bioanalyzer. All RNA samples were quantified and qualified with an
RNA integrity number 48. RNA-seq libraries were constructed following the Illu-
mina kit recommendations. The constructed libraries, indexed with six nucleotide
sequences (barcode), were pooled together with equimolar amounts (2 nM) and were
sequenced on the Illumina HiSeq 2000 sequencer with 2 100 bp. Raw data have
been deposited in GenBank under the accession PRJNA265955. The raw FASTQ
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10456 ARTICLE
NATURE COMMUNICATIONS | 7:10456 | DOI: 10.1038/ncomms10456 | www.nature.com/naturecommunications 7
format data generated from CASAVA v1.8.2 were first assessed for quality using
FASTQC v0.10.1 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and
FASTX toolkit v0.0.13.2 (http://hannonlab.cshl.edu/fastx_toolkit/). Poor quality
reads (Phred scoreo20) were trimmed at both ends with SolexaQA packages v2.2
(http://solexaqa.sourceforge.net/); only the reads with lengths Z25 bp on both sides
of the paired-end format were subjected to further analysis. The data were then
aligned with the G. hirsutum (TM-1) genome (PRJNA248163). The software
Cufflinks v2.2.1 (http://cufflinks.cbcb.umd.edu/) was used to accurately quantify the
abundance of genes and calculate the fragments per kilobase of genes per million
mapped reads (FPKM). Differential expression was defined as a gene with a
minimum of a twofold change (TM-1 versus PGF-silenced TM-1 and TM-1
versus Hai-1) with RPKM41ineitherTM-1orPGF-silenced TM-1 or Hai-1.
K means clustering was performed with the open-source program, Cluster3.0
(http://bonsai.hgc.jp/Bmdehoon/software/cluster/software.htm). The genes in each
cluster were then classified into MapMan functional categories40. Changes in the
significance of expression were investigated in functional categories of the MapMan
annotation through the application of a two-sided Wilcoxon rank test with a
Benjamini-Yekutieli correction for multiple tests. Pathway analysis was mainly based
on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database41.
Development of the CYP706B1 RNAi transgenic line.To unravel the
relationship between gossypol and glands, we cultivated the CYP706B1 RNAi
transgenic lines. A 408 bp fragment of CYP706B1 (GenBank: AF332974.1) was
amplified from the leaf cDNA library with dsP1-F-BamHI: 50-GGATCCTCAGC
TCGTATTCATGGCTG-30and dsP1-R-XbaI: 50-TCTAGACAAATACAATATC
ATTGAGG-30primers, and dsP1-F-SacI: 50-GAGCTCTCAGCTCGTATTCATGG
CTG-30and dsP1-R-NotI: 50-GCGGCCGCCAAATACAATATCATTGAGG-30
primers. The dsRNA construct was inserted into the BamHI-SacI site of binary
vector pCAMBIA2301. The 1,143 bp globulin promoter, Proglobulin, was amplified
with PG-F-HindIII: 50-AAGCTTCTATTTTCATCCTATTTAGA-30and
PG-R-BamHI: 50-GGATCCGATTACGATAAGCTCTGTAT-30primers from
cotton genomic DNA and inserted to the HindIII/BamHI site to control dsRNA
construct expression. The resultant constructs of Proglobulin::dsCYP706B1 (P1)
was transferred into A. tumefaciens strain LBA4404 and then used for cotton
transformation. Cotton seeds (R15) were surface-sterilized with 70% ethanol for
1 min, and 15% H
2
O
2
for 4 h, followed by washing with sterile water five times. The
sterilized seeds were germinated on Murashige and Skoog (MS) medium under
dark conditions at 28 °C for 7 days. Hypocotyls were cut into 1-cm fragments and
incubated with the overnight culture of Agrobacterium for 20 min. After 2 days of
co-cultivation, hypocotyl explants were transferred to MS medium containing
1mgml12,4-Dichlorophenoxyacetic acid (2,4-D), 50 mgml1kanamycin and
400 mgml1cefalotin for 2 months. The resistant calli were transferred to
hormone- and antibiotic-free MS medium for somatic embryogenesis and plant
regeneration. The resultant transgenic plants were transferred into soil for growing.
Seeds from transgenic line P1-13-8 and progeny plants showed low gossypol
contents. Genomic DNA from P1-13-8 and R15 were prepared for Southern blot
analysis. HindIII- or EcoRI-digested genomic DNAs were separated on a 1%
agarose gel, transferred to a Hybond-Nþnylon membrane (GE Healthcare Life
Sciences) and probed with NPTII gene. The DIG High Primer DNA Labeling and
Detection Starter kit II (Roche Applied Science, USA) were used for labelling and
hybridization according to the manufacturer’s protocol.
Yeast one-hybrid assay.The yeast one-hybrid assay was performed using the
MATCHMAKER one-hybrid system (Clontech). Fragments containing four tan-
dem copies of G-box (50-CACGTG-30)(4G-box WT) and G-box mutant
(50-CATAGA-30;4G-box mutant) were synthesized by GenScript Biotechnology
Co., Ltd. These two fragments were ligated into the HindIII-XhoI sites of pAbAi.
The bait constructs were linearized with BstBI and integrated into the yeast genome
(strain Y1H). Various concentrations of aureobasidin A (AbA; Clontech, cat. no.
630446) on SD-Ura medium were used to identify the basal expression of AUR1-C.
The ORF of GoGPF was ligated to the GAL4 activation domain in pGAD424. Yeast
transformants were tested on SD/-Ura medium containing 20 ng ml 1AbA.
Phylogenetic analysis.Alignment of the amino acid sequences of the GoPGF and
bHLH families in other species was performed using the CLUSTALX program42.A
neighbour-joining (NJ) method was then applied to produce a phylogenetic tree.
The relative degree of branch support was determined within the NJ framework
using the bootstrap procedure. The original data set was resampled 1,000 times.
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Acknowledgements
This work was financially supported in part by grants from NSFC(31201250) the Major
State Basic Research Development Program of China (973 Program, 2011CB109300), the
Priority Academic Program Development of Jiangsu Higher Education Institutions and
the 111 Program.
Author contributions
T.Z. and X.C. conceptualized the research programme. T.Z., X.C. and Y.H. designed
experiments and coordinated the project. D.M., Y.H., C.Y., B.L., Q.W., W.L., L.W., H.W.,
L.D., C.D., S.W., S.C., C.C., X.Z., X.G., B.Z., S.Z. and W.G. conducted laboratory
experiments, and were involved in data analysis. B.L., L.F., D.M., G.M., M.P. and J.C.
performed bioinformatic analyses. D.M., T.Z., Y.H., X.C., L.F. and J.W. participated in
preparing and revising the manuscript. T.Z. and X.C. supervised data generation and
analysis. All authors discussed results and commented on the manuscript.
Additional information
Accession codes: Sequences have been deposited at DDBJ/EMBL/GenBank under the
accessions KP072743 (GoPGF_A12 of TM-1), KP072744 (GoPGF_D12 of TM-1),
KP072745 (GoPGF_A12 of Hai-1) and KP072746 (GoPGF_D12 of Hai-1). Transcriptome
data have been deposited in GenBank under the accession PRJNA265955.
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing financial interests: The authors declare no competing financial interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Ma, D. et al. Genetic basis for glandular trichome formation in
cotton. Nat. Commun. 7:10456 doi: 10.1038/ncomms10456 (2016).
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