GhCWIN1
GhCWI1 (1)
(1) --------------------------------------MDISLFLFVGFSCFLLINGNKVEDCDYLTDKTSLQSFQVLLQAE------------------Atßfruct2 (1)
Atβfruct2
(1) -------------------------------------MSAPKFGYVLLLIVLINISNNG-VDAFHKVFKKLQSKSTSLESVS------------------Lin6
Lin6
NtCWIN1
NtCWIN1
OsCWIN1
OsCWI1
ZmCWIN1
ZmCWIN1
Atßfruct4
Atβfruct4
GhVIN1
GhVIN1
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
-------------------------------MEILRKSSSLWALPILVL--CFFINNGVFVDASHKVYMHLQSTTSHVDASK-------------------------------------------------MEFLR-NSSLWSLQILLLGVFVFVSNNGGVDASHKVYMHLQSTSSNVKN-------------------------------------------------MGTR--LLALAPW----LLLLLLQLAGASHVVHRSLEAEQAPSSVPASIVSP------------------------------------------------MGTRPRGVVLAPWAVVLVLVLALRLAGASHVIHRSLEAEAAP-SVPASIVSP-------------------DALLPISAREEEPLCPYTRLPMADPNQETHGPRRRRPFKGLLAVSFGLLFIAFYVALIATHDG----SRSNDEGIDETETITSRARLAGVSEKRNDGLWKL
-----MEASSSTSHDPALFHAPLLHHPRRRSSRPLKGFAVIFGSVVFLLSLVILIVNQSPEPLASNPSSVTEAGSYSMAAQP-RGIAEGVSAKSNPSLFDK
GhCWIN1
GhCWI1 (45)
(45) --------Q--------PYRTAYHFQSPQDWLNDPNGPMYYKGVYHLFYQYNPNSAWFGDGMVWAHSASYDLINWFGLDHALVPSEPFDAISCWSGSATIL
Atßfruct2 (45)
Atβfruct2
(45) --------P--------LHRTAYHFQPPRHWINDPNAPMLYKGVYHLFYQYNPKGAVWGN-IVWAHSVSKDLINWEALEPAIYPSKWFDINGTWSGSATHV
Lin6
Lin6 (50)
(50)
NtCWIN1
NtCWIN1 (49)
(49)
OsCWIN1
OsCWI1 (47)
(47)
ZmCWIN1
ZmCWIN1 (52)
(52)
Atßfruct4 (98)
Atβfruct4
(98)
GhVIN1
GhVIN1 (96)
(96)
-----------------VHRTGYHFQPPKNWINDPNGPMYYNGVYHLFYQYNPKGATWGN-IVWAHSVSKDLINWIPLEPAIYPSKVFDKYGTWSGSATIL
GhCWIN1
(130)
GhCWI1(130)
Atßfruct2 (129)
Atβfruct2(129)
Lin6
(133)
Lin6(133)
NtCWIN1
(132)
NtCWIN1(132)
OsCWIN1
(130)
OsCWI1(130)
ZmCWIN1
(135)
ZmCWIN1(135)
Atßfruct1 (198)
Atβfruct4(198)
GhVIN1
(190)
GhVIN1(190)
PGDKPVILYTGIDAN--YNQVQNLATPKNESDPLLVEWVKY-FGNPLMTPPDG-VKGDNFRDPTTAWQG-PDGTWKVVIGSYSNNQ-GMAILFQS-QDFVH
GhCWIN1
(224)
GhCWI1(224)
Atßfruct2 (224)
Atβfruct2(224)
Lin6
(229)
Lin6(229)
NtCWIN1
(227)
NtCWIN1(227)
OsCWIN1
(226)
OsCWI1(226)
ZmCWIN1
(232)
ZmCWIN1(232)
Atßfruct1 (292)
Atβfruct4(292)
GhVIN1
(285)
GhVIN1(285)
WTMHRDPLYSSLKTEMWECPDFFPVSIN-ST-NGVDTSVE-NPSVRHVMKASFNS--HDYYTVGTYVTEQERFLPDADFTGTSLDL---RFDYGKFYASKS
GhCWIN1
(317)
GhCWI1(317)
Atßfruct2 (320)
Atβfruct2(320)
Lin6
(324)
Lin6(324)
NtCWIN1
(322)
NtCWIN1(322)
OsCWIN1
(318)
OsCWI1(318)
ZmCWIN1
(326)
ZmCWIN1(326)
Atßfruct1 (389)
Atβfruct4(389)
GhVIN1
(380)
GhVIN1(380)
FFDGKKNRRILCAWVNESDIMEDDLXKGWSGLQSIPRQIWLDL-XGKQLVQWPVEELNSLRGNEVYVYDKQLESGSVFEVSGITASQADIEIMSELPK---
GhCWIN1
(414)
GhCWI1(414)
Atßfruct2 (417)
Atβfruct2(417)
Lin6
(421)
Lin6(421)
NtCWIN1
(419)
NtCWIN1(419)
OsCWIN1
(415)
OsCWI1(415)
ZmCWIN1
(426)
ZmCWIN1(426)
Atßfruct1 (487)
Atβfruct4(487)
GhVIN1
(479)
GhVIN1(479)
---LEEAEFIDTSLVDP--QLICDKQDASVTGKFGPFGLLALATKDLTEQTAIFFRVFR---DHKDYKVLMCSDQKRSSLRNELD--------KTTYGAFI
GhCWIN1
(499)
GhCWI1(499)
Atßfruct2 (513)
Atβfruct2(513)
Lin6
(510)
Lin6(510)
NtCWIN1
(508)
NtCWIN1(508)
OsCWIN1
(503)
OsCWI1(503)
ZmCWIN1
(515)
ZmCWIN1(516)
Atßfruct1 (575)
Atβfruct4(576)
GhVIN1
(558)
GhVIN1(558)
-----------------VHRTGYHFQPPKNWINDPNGPMYYNGVYHLFYQYNPKGAVWGN-IVWAHSVSTDLINWIPLEPAIYPSKIFDKFGTWSGSATIL
-----------------LLRTGYHFQPPMNWINDPNGPLYYKGWYHLFYQYNPKGAVWGN-IVWAHSVSQDLINWIALEPAIKPDIPSDQYGCWSGSATIL
-----------------LLRTGYHFQPPMNWINDPNAPLYYKGWYHLFYQYNPKGAVWGN-IVWAHSVSRDLINWVALEPAIYPSIPSDKYGCWSGSATIL
SGDRNTPAFEWNNSMLSWQRTAFHFQPEQNWMNDPNGPLFYKGWYHFFYQYNPNAAVWGD-IVWGHAVSRDLIHWVHLPIAMVADQWYDSNGVWTGSATFL
VGF--N----WTNAMFYWQRTAYHFQPQKNWMNDPDGPLYHKGWYHLFYQYNPDSAIWGN-ITWGHAVSKDLIHWFYLPLAMVPDQWYDINGCWTGSATLL
PGKGPVILYTGITEN--QTQIQNYAIPQDLSDPYLKTWIKP-DDNPIVKPDNG-ENGSAFRDPTTAWFNKKDGYWRMLVGSKRKNR-GIAYMYKS-RDFKK
PGNKPVILYTGIVDAN-KTQVQNYAIPANMSDPYLRKWIKP-DNNPLIVADKN-INKIQFRDPTTAWMG-RDGYWRVLVGSVRNHR-GKVIMYKSNKNFMK
PGNKPIILYTGIIDAN-RTQVQNYAIPANLSDPYLRKWIKP-DNNPLIVADMS-INKTQFRDPTTAWMG-RDGHWRILVGSVKNHR-GKVILYRS-KDFMK
PDGTPAILYTGIDRPNINYQVQNIAFPKNASDPLLREWVKP-AYNPVATPEPG-MNATQFRDPTTAWYA--DGHWRMLVGGLKGARLGLAYLYRS-RDFKT
EDGTPAILYTGIDRADINYQVQVLALPKDASDPLLREWEKPEEYNPVATPAAGGINATQFRDPTTAWRH--AGHWRMLVGSVRGAR-GMALVYRS-RDFRK
PDGSIVMLYTGSTDKA--VQVQNLAYPEDPNDPLLLKWVKF-PGNPVLVPPPG-ILPKDFRDPTTAWKTSEG-KWRITIGSKLN-KTGISLVYDT-IDFKT
PDGRIVMLYTGSTNES--VQVQNLAYPANLSDPLLLQWLKY-PGNPVVVPPTG-IEDNEFRDPTTAWLGPDG-SWRITVGTRFNTTIGTALVFQT-TNFSD
WVKSKRPIHSRKKTGMWECPDFFPVSVT-DKKNRLDFSYD-GPNAKHVLKVSLDLTRYEYYTLGTYDTKKDRYRPDGYTPDGWDGL---RFDYGNYYASKT
WTKAKHPLHSAQGTGNWECPDFFPVSLK-NE-NGLDTSYD-GKDVKHVLKVSFDVTRFDHYTVGTYDTKKDKYFPDNTSIDGWKGL---RLDYGNYYASKA
WTKAKHPLHTATNTGNWECPDFFPVSLK-HT-NGLDTSYR-GEYTKHVLKVSLDVTRFEYYTVGTYDTRKDRYIPDNTSVDGWKGL---RLDYGNYYASKS
WVRAKHPLHS-ALTGMWECPDFFPLQAP-GLQAGLDTSVP---SSKYVLKNSLDLTRYDYYTVGIYNKVTERYVPDNPAGDYHRLR----YDYGNFYASKT
WTKAKHPLHSAALTGMWECPDFFPVSGP-GLQAGLDTSAP---GTKYVLKSSLDLTRYDYYTIGSYDGGKDRYYPDDPAGDYHHRR---RYDYGNYYASKT
YEKLDTLLHRVPNTGMWECVDFYPVSKT--AGNGLDTSVN-GPDVKHIVKASMDDTRFDHYAVGTYFDSNGTWIPDDPTIDVGMTASLR-YDYGKFYASKS
YQLLDGVLHAVPGTGMWECVDFYPVAIN--GSVGLDTTAL-GPGIKHVLKASLDDTKVDHYAIGTYDMITDKWTPDNPEEDVGIG--LK-VDYGRYYASKT
FFDDKTNRRILWGWANESDTVQDDTVKGWAGIQLIPRTILLDS-SGKQLVFWPIEEIESLRGKNVQMTNQKMEMGQRFEVQGITPAQVDVDVTFNVGN--FFDSGKNRRILLGWANESDTVDNDVKKGWAGVHPIPRKIWLDP-SGKQLVQWPVQELETLRKKKVQLNNKKLNKGEKVEIKGITVAQADVEVIFSFAS--FFDPSKNRRIMLGWANESDTVDDDVRKGWAGVHPIPRKLWLDP-SGKQLVQWPVEELETLRKKKVQLSNHKLYKGEMIEVKGITVAQADVEVTFSFAS--FFDPVKHRRILLGWANESDSVTYDKAKGWAGIHAIPRKVWLDP-SGKQLLQWPIEELETLRGKSVSVFDKVVKPGEHFQVTGLGTYQADVEVSLEVSG--FYDPVERRRVLLGWANESDSVPDDKAKGWAGIHAIPRKIWLDP-TGKQLLQWPIHEVEKLRGKAVSVDAKLVKPGDHFEVTGIATYQADVEVSFELELEAG
FYDQNKGRRVLWSWIGESDSEASDVQKGWSSLQGIPRTVVLDTKTGKNLVQWPVEEIKSLRLSSKQFD-LEVGPGSVVPVDVGSAAQLDIEAEFEINKE-FFDQSKQRRILYGWVNETDTEADDLEKGWASIQTIPRSVLYDNKTGTHLLQWPVEEVESLRLNATVFKDVVVEAGSVVPLDIGTATQLDILAEFEIETL--
---LEKAEKFDESFATK-PLELCNLKGSNVNGGVGPFGLITLATSDLEEYTPVFFRVFKDAASN-KPKVLMCSDAKPSSLKKDTGTDAKERMYKPSFAGFV
---LDKAEPFDSSWADLYAQDVCAIKGSTVQGGLGPFGLLTLASKNLEEYTPVFFRVFK--AHD-NYKVLMCSDASRSSLKNET------TMYKPSFAGYV
---LDKAEPFDPSWADLYAQDVCAIKGSTVQGGRGPFGLITLASKNLEEYTLVFFRVFK--AQD-KYKVLMCSDASRSTLKNET------TMYKPSFGGYV
---LEKAEALDPAFGDN-AERLCGAKGADVRGGV-VFGLWVLASAGLEEKTAVFFRVFKPAGHGAEPVVLMCTDPTKSSLSPDL-------Y-KPTFAGFV
TSLLEKAEAFDPAYDDD-AQKLCGVKGADARGGVGPFGLWVLASADLQERTAVFFRVFRDGHG--KPKVLMCTDPTKSSLSPDL-------Y-KPTFAGFV
--SLDKIIGNASVVAEAEEFSCEKSGGSTVRGALGPFGFSVLATESLSEQTPVYFYVAK--GKDSELKTFFCTDTSRSSVANDVV--------KPIYGSVV
--VSNSTED-----EVSDCGDG-----AVDRNTYGPFGVLVIADDSLSELTPIYFRPLN--ISDGSLETYFCADETRSSKAPNVT--------KRVYGGKV
DIDPQREMVSLRTLI------DHSIIESFGGKGRSVITTRVYPKLAIYDEAHLFAFNNG-SLSVTVSRLNAWSMNKAQITAGGKTETISEDFSRKK
DVGLLDGKISLRSLI------DHSVVESFGAKGKTVITSRVYPTKAVGEKAHLFVFNNG-SQPVTVESLNAWNMQKPLKMNQGAK----------DVDLADKKLSLRSLI------DNSIVESFGAGGKTCITPRVYPTLAIFDKAHLFAFNNG-AETITIETLNAWSMANAKLH---------------DVDLADKKLSLRSLI------DNSVVESFGAGGKTCITSRVYPTLAIFDKAHLFAFNNG-TEAITIETLNAWSMADAKLH---------------DTDISSGKISLRSLI------DRSVVESFGAGGKTCILSRVYPSMAIGDKAHLYVFNNG-EADIKISHLKAWEMKKPLMNGA-------------DADISSGKITLRSLI------DRSVVESFGAGGKTCILSRVYPSIAVGKDAHLYVFNNG-EVDVTVSGLTAWEMKKPLMNGA-------------PVLK-GEKLTMRILV------DHSIVEAFGQGGRTCITSRVYPTTAIYGAAKLFLFNNALDATVTAS-FTVWQMNSAFIHPYSDEAVRALSRT--PVLD-DENYNMRVLV------DHSVVESFGEGGRTVITSRVYPTEAIYGAARLFLFNNASRVNVKAT-LKIWEMNSAFIRPFPFEETLFQEMVAST
Supplemental Figure 1
Supplemental Figure 1. Alignment of GhCWIN1 sequence with five plant CWINs (Atβfruct2,
Lin6, NtCWIN1, OsCWIN1 and ZmCWIN1) and two VINs (Atβfruct4 and GhVIN1). Thirteen
boxed sequences indicate the conserved regions of plant invertases. The β-fructosidase motif
(NDPD/NG) and cysteine catalytic domain (WECP/VD) were underlined. Arrowsheads denote
five consistently different amino acids between VINs and CWINs. Arrow show GhCWIN1 signal
sequence cleavage site between residue 19 and residue 20.
Plant CIN
Plant VIN
Bacterial
invertase
Yeast
invertase
Plant CWIN
0.1
Supplemental Figure 2
Supplemental Figure 2. Phylogenetic analysis classifies GhCWIN1 as a plant CWIN. Genbank
accession number for each invetrase listed and the corresponding species were listed in
Supplemental Table 1.
Seed
5
Filial tissue
10
15
20
DAA
GhCWIN1
GhINH1
GhMST1
GhSUT1
Gh18srRNA
Supplemental Figure 3
Supplemental Figure 3. Transcript level of cotton cell wall invertase GhCWIN1, invertase
inhibitor GhINH1, monosaccharide tranporter GhMST1, sucrose transporter GhSUT1 in cotton
seed and filial tissue at different development stages. Gh18srRNA was used as an internal
control.
25
25
a
20
20
b
b
15
15
c
10
10
55
00
5
10 15 20
(mg/g
content
Glucose
g-1FW)
(mg
content
Glucose
FW)
(mg/g
content
Glucose
g-1FW)
(mg
content
Glucose
FW)
A
25
25
a
20
20
b
b
15
15
c
10
10
55
00
Seed Seed coat
5
10 15 20
Seed Filial tissue
a
20
20
ab
15
15
b
b
10
10
55
00
5
10 15 20
Fructose content (mg/g FW)
25
25
Fructose content (mg g-1 FW)
Fructose content (mg/g FW)
Fructose content (mg g-1 FW)
B
25
25
a
20
20
b
15
15
bc
c
10
10
55
00
Seed Seed coat
5
10 15 20
Seed Filial tissue
25
25
20
20
15
15
10
10
55
00
5
10 15 20
Seed Seed coat
Sucrose content (mg/g-1FW)
content (mg g FW)
Sucrose
Sucrose content (mg/g -1
content (mg g FW)
Sucrose
FW)
C
25
25
a
20
20
15
15
ab
bc
10
10
c
55
00
5
10 15 20
Seed Filial tissue
Supplemental Figure 4
Supplemental Figure 4. Glucose (A), fructose (B) and sucrose (C) level in cotton seed, seed
coat and filial tissue during development. Each value is the mean ± SE of at least three
biological replicates. Different letters indicate significant differences at P ≤.05 according to
randomization one-way ANOVA test.