(A)
(B)
Xu142
-1
0
1
Xu142 fuzzless-lintless mutant
-1
0
1 DAA
Control
Activity
in seed
surface
Magnified
view
(C)
Enzyme activity
(μmol mg protein-1 min-1)
2.0
1.5
CIN
VIN
CWIN
1.0
0.5
0
-1
0
Xu-142
1
-1
0
1 DAA
Xu-142 fl mutant
Supplemental Figure 1
Supplymental Figure1. In situ localization and assay of invertase activity in a fiberless cotton
seed mutant and its wild-type.
(A) Histochemical staining of invertase activity in the wild type cotton (G. hirsutum L. cv Xu142) ovule at -1 DAA and seed at 0- and 1-DAA. Note, strong invertase activity signals indicated
by the dark blue color in the seed surface at 0 and 1 DAA but its absence at -1 DAA before fiber
initiation. A magnified view was presented from the boxed region. Also note the absence of the
signals in the negative controls.
(B) Histochemical staining of invertase activity in a fiberless mutant from the G. hirsutum L. cv.
Xu-142 background. Note, no invertase activity was localized at the seed surface. Bars = 1 mm in
(A) and (B).
(C) Measurement of invertase activity from extract of ovule and seed described in (A) and (B).
Note the significantly higher activity of VIN but not CWIN and CIN in the wild-type seed than
that of the fiberless mutant. Also note, in the wild type the VIN activity was higher at 0 and 1
DAA than that in ovule at -1DAA, consistent with observation from the histochemical staining in
(A). Each value is the mean of six biological replicates with SE.
GhVIN1
(1) -------MEASSSTSHDPALFHAPLLHHPRR------RSSRPLKGFAVIFGSVVFLLSLVILIVNQSPEPLASNPSSVTEAGSYSMAAQPRGIAEGVSAK
LeVIN1(Z12025)
(1) -------MATQCYDPENSASRYTLLPDQPDS------GHRKSLKIISGIFLSVFLLLSVAFFPILN-------NQSPDLQIDSRSPAPPSRGVSQGVSDK
OsINV3(AF276704)
(1) -----METRDDVADASALPYSYSPLPAGDAASADLAAARRSRRRPLCVALFLASAAVILAVAVLSGVRLAGRPATTTMVVPGVVEMEMASRGPESGVSEK
ZmIvr1(U16123)
(1) MIPAVADPTTLDGGGARRPLLPETDPRGRAAAGAEQKRPPATPTVLTAVVSAVLLLVLVAVTVLASQHVDGQAGGVPAGEDAVVVEVAASRGVAEGVSEK
OsCWI1(AB073749)
(1) ------------------------------------------------MGTRLLALAPWLLLLLLQ------------LAGASHVVHRSLEAEQAPSSVP
NtCWIN1(X81834)
(1) ------------------------------------------MEFLRNSSLWSLQILLLGVFVFVS------------NNGGVDASHKVYMHLQSTSSNV
GhVIN1
(88) SN----------PSLFDKVGFNWTNAMFYWQRTAYHFQPQKNWMNDPDGPLYHKGWYHLFYQYNPDSAIWGN-ITWGHAVSKDLIHWFYLPLAMVPDQWY
LeVIN1(Z12025)
(81) TFR--------DVAGASHVSYAWSNAMLSWQRTAYHFQPQKNWMNDPNGPLYHKGWYHLFYQYNPDSAIWGN-ITWGHAVSKDLIHWLYLPFAMVPDQWY
OsINV3(AF276704)
(96) TSGAEEMVRLMG-GAAGGEAFPWSNAMLQWQRTGFHFQPERNWMNDPNGPVYYKGWYHLFYQYNPDGAVWGNKIAWGHAVSRDLVHWRHLPLAMVPDQWY
ZmIvr1(U16123) (101) -S------TAPLLGSGALQDFSWTNAMLAWQRTAFHFQPPKNWMNDPNGPLYHKGWYHLFYQWNPDSAVWGN-ITWGHAVSRDLLHWLHLPLAMVPDHPY
OsCWI1(AB073749)
(41) AS-----------------------IVSPLLRTGYHFQPPMNWINDPNGPLYYKGWYHLFYQYNPKGAVW-GNIVWAHSVSQDLINWIALEPAIKPDIPS
NtCWIN1(X81834)
(47) K--------------------------NV-HRTGYHFQPPKNWINDPNGPMYYNGVYHLFYQYNPKGAVW-GNIVWAHSVSTDLINWIPLEPAIYPSKIF
GhVIN1
(177) DINGCWTGSATLLPDGRIVMLYTGSTNES--VQVQNLAYPANLSDPLLLQWLKYPGNPVVVPPTG--IEDNEFRDPTTAWLGP---DGSWRITVGTRFNT
LeVIN1(Z12025) (172) DINGVWTGSATILPDGQIMMLYTGDTDDY--VQVQNLAYPANLSDPLLLDWVKFKGNPVLVPPPG--IGVKDFRDPTTAWTGPQ--NGQWLLTIGSKIGOsINV3(AF276704) (195) DVNGVWTGSATTLPDGRLAMLYTGSTNAS--VQVQCLAVPSDPDDPLLTNWTKYHANPVLYPPRT--IGDRDFRDPTTAWRDPS--DGDWRIVIGSKDEH
ZmIvr1(U16123) (193) DANGVWSGSATRLPDGRIVMLYTGSTAESS-AQVQNLAEPADASDPLLREWVKSDANPVLVPPPG--IGPTDFRDPTTACRTPAGNDTAWRVAIGSKDRD
OsCWI1(AB073749) (117) DQYGCWSGSATILPDGTPAILYTGIDRPNINYQVQNIAFPKNASDPLLREWVKPAYNPVATPEPG--MNATQFRDPTTAWY----ADGHWRMLVGGLKGA
NtCWIN1(X81834) (119) DKFGTWSGSATILPGNKPIILYTGIIDANR-TQVQNYAIPANLSDPYLRKWIKPDNNPLIVADMS--INKTQFRDPTTAWMG---RDGHWRILVGSVK-N
GhVIN1 (270) TIGTALVFQTTNFSDYQLLD-GVLHAVPGTGMWECVDFYPVAIN------GSVGLDTTAL-GPGIKHVLKASLDDTKVDHYAIGTYDMITDKWTPDNPEE
LeVIN1(Z12025) (265) KTGVALVYETSNFTSFKLLD-GVLHAVPGTGMWECVDFYPVSTK------KTNGLDTSYN-GPGVKHVLKASLDDNKQDHYAIGTYDLGKNKWTPDNPEL
OsINV3(AF276704) (289) HAGIAVVYRTADFVTYDLLP-GLLHRVEATGMWECIDFYPVAGG--EGVDMTEAMYARN---KGVVHVMKASMDDDRHDYYALGRYDPARNAWTPLDAAA
ZmIvr1(U16123) (290) HAGLALVYRTEDFVRYDPAP-ALMHAVPGTGMWECVDFYPVAAGSGAAAGSGDGLETSAAPGPGVKHVLKASLDDDKHDYYAIGTYDPATDTWTPDSAED
OsCWI1(AB073749) (211) RLGLAYLYRSRDFKTWVRA-KHPLHSAL-TGMWECPDFFPLQAPGLQA-----GLDTSVPS---SKYVLKNSLDLTRYDYYTVGIYNKVTERYVPDN--NtCWIN1(X81834) (212) HRGKVILYRSKDFMKWTKA-KHPLHTATNTGNWECPDFFPVSLKHTN------GLDTSYRG-EYTKHVLKVSLDVTRFEYYTVGTYDTRKDRYIPDN--T
GhVIN1 (362) DVGIG--LKVDYGRYYASKTFFDQSKQRRILYGWVNETDTEADDLEKGWASIQTIPRSVLYDNKTGTHLLQWPVEEVESLRLNATV-FKDVVVEAGSVVP
LeVIN1(Z12025) (357) DCGIG--LRLDYGKYYASKTFYDPKKERRVLWGWIGETDSESADLQKGWASVQSIPRTVLYDKKTGTHLLQWPVEEIESLRVGDPT-VKQVDLQPGSIEL
OsINV3(AF276704) (383) DVGIG--LRYDWGKFYASKTFYDPAKRRRVLWGWVGETDSERADVAKGWASLQ-------LDTKTGSNLLQWPVEEVETLRTNSTD-FGGITVDYASVFP
ZmIvr1(U16123) (389) DVGIG--LRYDYGKYYASKTFYDPVLRRRVLWGWVGETDSERADILKGWASVQSIPRTVLLDTKTGSNLLQWPVVEVENLRMSGKS-FDGVALDRGSVVP
OsCWI1(AB073749) (298) PAGDYHRLRYDYGNFYASKTFFDPVKHRRILLGWANESDSVTYDKAKGWAGIHAIPRKVWLDPS-GKQLLQWPIEELETLRGKSVS-VFDKVVKPGEHFQ
NtCWIN1(X81834) (302) SVDGWKGLRLDYGNYYASKSFFDPSKNRRIMLGWANESDTVDDDVRKGWAGVHPIPRKLWLDPS-GKQLVQWPVEELETLRKKKVQLSNHKLYKG-EMIE
GhVIN1 (544) KAPNVT--KRVYGGKVPVLDD-ENYNMRVLVDHSVVESFGEGGRTVITSRVYPTEAIYGAARLFLFNNASRVNVKAT-LKIWEMNSAFIRPFPFEETLFQ
LeVIN1(Z12025) (544) EAPGVG--KQVYGSSVPVLDG-EKHSMRLLVDHSIVESFAQGGRTVITSRIYPTKAVNGAARLFVFNNATGASVTAS-VKIWSLESANIQSFPLQDL--OsINV3(AF276704) (565) HADDIV--KRVVGNVVPVLDG-ETFSLRVLVDHSIVESFAQGGRSTATSRVYPTEAIYANAGVFLFNNATSARVTAKKLVVHEMDSSYNQAYMA-----ZmIvr1(U16123) (575) KANDLV--KRVYGSLVPVLDG-ENLSVRILVDHSIVESFAQGGRTCITSRVYPTRAIYDSARVFLFNNATHAHVKAKSVKIWQLNSAYIRPYPATTTSLOsCWI1(AB073749) (489) LSPDL--YKPTFAGFVDTDISSGKISLRSLIDRSVVESFGAGGKTCILSRVYPSMAIGDKAHLYVFNNG-EADIKISHLKAWEMKKPLMNGA-------NtCWIN1(X81834) (492) LKNETTMYKPSFGGYVDVDLADKKLSLRSLIDNSVVESFGAGGKTCITSRVYPTLAIFDKAHLFAFNNG-TEAITIETLNAWSMADAKLH----------
Supplemental Figure 2
Supplemental Figure 2. Alignment of GhVIN1 sequence with three plant VINs (LeVIN1,
OsINV3 and ZmIvr1 and two CWINs (OsCWI1 and NtCWIN1).
Thirteen boxed sequences indicate the conserved regions of plant invertases. The β-fructosidase
motif (NDPD/NG) and cysteine catalytic domain (WECP/VD) were underlined. Arrows denote
five consistently different amino acids between VINs and CWINs. The “LL” or “PLP” site
(double underline), strongly basic region (dot line) hydrophobic transmembrane segment (dash
line) and a dashed-box representing motifs characteristic of VINs were marked on the Nterminus of the VINs including GhVIN1.
Plant CIN
Plant VIN
Bacterial
invertase
Plant CWIN
Yeast
invertase
0.1
Supplemental Figure3. Phylogenetic analysis classifies GhVIN1 as a VIN.
Genbank accession number for each invetrase listed and the corresponding species were listed in
Supplemental Table 1. Calculated pI values are indicated in brackets.
(B)
OE
WT
Root length (mm)
(A)
null 2-10 5-8
Atβfruct4
GhVIN1
AtTUB
WT
Root cell length
(μm)
(C)
A
190
B
170 BC
C
150
null
OE 2-10
50
45
B
35
(D)
100%
Root hair
80%
length (μm)
> 400
201-400
1-200
(G)
(F)
20%
0%
(H)
Enzyme activity
(μmol mg protein-1 min-1)
(E)
B
OE 5-8
40%
WT: 149.03±2.62 μm (116)
OE null: 146.99 ±2.58 μm(113)
OE 2-10: 158.71 ±6.5 μm (118)
OE 5-8: 179.17 ±4.22 μm (116)
A
40
60%
130
A
VIN
CWIN
2.5
2.2
A
B
B
A
2.5
A
A
A
A
2.2
1.9
1.9
1.6
1.6
Supplemental Figure 4
Supplemental Figure 4. Expression of GhVIN1 in wild-type Arabidopsis promotes root cell
elongation.
(A) Semi-quantitative RT-PCR analysis confirmed the over-expression (OE) of GhVIN1 mRNA
in line 2-10 and 5-8. AtTUB was used as an internal control.
(B) GhVIN1 OE lines 2-10 and 5-8 displayed a longer-root phenotype, in comparison with that of
WT and OE null.
(C) Increased cell length in GhVIN1 OE lines of 2-10 and 5-8, as compared with WT and OE null
plants.
(D) Percentage of different sized root hairs in Arabidopsis Atβfruct4 T-DNA insertion mutants
(inv-5 and -7), GhVIN1 complementation (C) lines (null, C7 3-2 and C14 2-1), wild type (WT)
and GhVIN1 over-expression (OE) lines (OE null, OE 2-10 and OE 5-8). Each value is the mean
of 120 root hairs derived from 10 seedlings.
(E) Histochemical staining of invertase activity in a root hair from GhVIN1 over-expression line
5-8, showing stronger signals (arrow)
(F) A negative control for (B) where sucrose was omitted from the reaction solution.
(G) A magnified view of a root hair from (B), showing invertase activity in vacuole (arrow) but
not in cell wall and cytoplasm (arrow head).
(H) Over-expression of GhVIN1 (line 2-10 and 5-8) increased VIN, but not CWIN, activities in
comparison with that of WT and OE null.
Different letters in (B), (C) and (H) indicate a significant difference at p < 0.01, according to
randomization one-way ANOVA test.
Bars = 1 mm in (B), 50 µm in (E) and 20 µm in (G). The scale in (F) is the same as that in (E).
Glucose
Sugar level
(mg g-1 dry weight)
4.0
A
4.0
Fructose
4.0
Sucrose
A
3.0
2.0
3.0
B
C
2.0
3.0
B
2.0
A
A
B
C
1.0
1.0
1.0
0
0
0
Supplemental Figure 5 Sugar levels in mature leaves of Arabidopsis inv-7 mutant, wild-type and
over-expression line OE 5-8.
Each value is the mean ± SE of three biological replicates
Different letters indicate a significant difference at p < 0.01 according to randomization one-way
ANOVA test.
Supplemental Figure 5