2.0 1.5 1.0 0.5 0 Enzyme activity (μmol mg protein-1 min -1 ) -1 0 1 -1 0 1 DAA Xu-142 Xu-142 fl...

8
2.0 1.5 1.0 0.5 0 Enzyme activity (μmol mg protein-1 m in -1 ) -1 0 1 -1 0 1 DAA Xu-142 Xu-142 fl mutant CIN VIN CWIN (C) Supplemental Figure 1 Control Activity in seed surface Magnified view (A) (B) 0 1 -1 0 Xu142 Xu142 fuzzless-lintless mutant

Transcript of 2.0 1.5 1.0 0.5 0 Enzyme activity (μmol mg protein-1 min -1 ) -1 0 1 -1 0 1 DAA Xu-142 Xu-142 fl...

Page 1: 2.0 1.5 1.0 0.5 0 Enzyme activity (μmol mg protein-1 min -1 ) -1 0 1 -1 0 1 DAA Xu-142 Xu-142 fl mutant CIN VIN CWIN (C) Supplemental Figure 1 Control.

2.0

1.5

1.0

0.5

0

Enz

yme

activ

ity

(

μmol

mg

prot

ein-

1 m

in-1)

-1 0 1 -1 0 1 DAA

Xu-142 Xu-142 fl mutant

CINVINCWIN

(C)

Supplemental Figure 1

Control

Activity in seed surface

Magnified view

(A) (B)

-1 0 1 -1 0 1 DAA

Xu142 Xu142 fuzzless-lintless mutant

Page 2: 2.0 1.5 1.0 0.5 0 Enzyme activity (μmol mg protein-1 min -1 ) -1 0 1 -1 0 1 DAA Xu-142 Xu-142 fl mutant CIN VIN CWIN (C) Supplemental Figure 1 Control.

Supplymental Figure1. In situ localization and assay of invertase activity in a fiberless cotton se

ed mutant and its wild-type.

(A) Histochemical staining of invertase activity in the wild type cotton (G. hirsutum L. cv Xu-14

2) 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 initi

ation. A magnified view was presented from the boxed region. Also note the absence of the signal

s 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). N

ote 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 th

an that in ovule at -1DAA, consistent with observation from the histochemical staining in (A). Ea

ch value is the mean of six biological replicates with SE.

Page 3: 2.0 1.5 1.0 0.5 0 Enzyme activity (μmol mg protein-1 min -1 ) -1 0 1 -1 0 1 DAA Xu-142 Xu-142 fl mutant CIN VIN CWIN (C) Supplemental Figure 1 Control.

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--NGQWLLTIGSKIG-

OsINV3(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-ENLSVRILVDHSIVESFAQGGRTCITSRVYPTRAIYDSARVFLFNNATHAHVKAKSVKIWQLNSAYIRPYPATTTSL-

OsCWI1(AB073749) (489) LSPDL--YKPTFAGFVDTDISSGKISLRSLIDRSVVESFGAGGKTCILSRVYPSMAIGDKAHLYVFNNG-EADIKISHLKAWEMKKPLMNGA--------

NtCWIN1(X81834) (492) LKNETTMYKPSFGGYVDVDLADKKLSLRSLIDNSVVESFGAGGKTCITSRVYPTLAIFDKAHLFAFNNG-TEAITIETLNAWSMADAKLH----------

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

Supplemental Figure 2

Page 4: 2.0 1.5 1.0 0.5 0 Enzyme activity (μmol mg protein-1 min -1 ) -1 0 1 -1 0 1 DAA Xu-142 Xu-142 fl mutant CIN VIN CWIN (C) Supplemental Figure 1 Control.

Supplemental Figure 2. Alignment of GhVIN1 sequence with three plant VINs (LeVIN1, OsIN

V3 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 f

ive consistently different amino acids between VINs and CWINs. The “LL” or “PLP” site (doubl

e underline), strongly basic region (dot line) hydrophobic transmembrane segment (dash line) an

d a dashed-box representing motifs characteristic of VINs were marked on the N-terminus of the

VINs including GhVIN1.

Page 5: 2.0 1.5 1.0 0.5 0 Enzyme activity (μmol mg protein-1 min -1 ) -1 0 1 -1 0 1 DAA Xu-142 Xu-142 fl mutant CIN VIN CWIN (C) Supplemental Figure 1 Control.

0.1

Bacterial invertase

Yeast invertase

Plant CINPlant VIN

Plant CWIN

Supplemental Figure3. Phylogenetic analysis classifies GhVIN1 as a VIN.

Genbank accession number for each invetrase listed and the corresponding species were listed in S

upplemental Table 1. Calculated pI values are indicated in brackets.

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(B)

(H)

Supplemental Figure 4

WT null OE 2-10 OE 5-8 (D)

C5

null

OE

null

0%

20%

40%

60%

80%

100%Root hair

length (μm)

> 400201-4001-200

inv-

5in

v-7

C7

3-2

C14

2-1

WT

OE

2-10

OE

5-8

Atβfruct4

GhVIN1

AtTUB

(A)

null 2-10 5-8

OEWT

35

40

45

50

Roo

t len

gth

(mm

)

OE

null

OE

2-10

OE

5-8

WT

BB

A A

(G)

(E)

(F) VIN

Enz

yme

activ

ity (

μmol

mg

prot

ein-1

min

-1) A A

BB

CWIN

1.6

1.9

2.2

2.5

WT

OE null

OE 2-10

OE 5-8

1.6

1.9

2.2

2.5

WT

OE null

OE 2-10

OE 5-8

A A

A A

(C)

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)

Roo

t cel

l len

gth

(μm

)

A

130

150

170

190

WT

OE

null

OE

2-10

OE

5-8

C

B BC

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Supplemental Figure 4. Expression of GhVIN1 in wild-type Arabidopsis promotes root cell elon

gation.

(A) Semi-quantitative RT-PCR analysis confirmed the over-expression (OE) of GhVIN1 mRNA i

n 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 (in

v-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 12

0 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 n

ot 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 co

mparison 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).

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Supplemental Figure 5

Glucose

0

1.0

2.0

3.0

4.0

WT

inv-

7O

E 5-

8

Suga

r le

vel

(mg

g-1

dry

wei

ght)

Fructose

0

1.0

2.0

3.0

4.0

WT

inv-

7O

E 5-

8

Sucrose

0

1.0

2.0

3.0

4.0

WT

inv-

7O

E 5-

8

B

C

A

B

C

A

B

A A

Supplemental Figure 5 Sugar levels in mature leaves of Arabidopsis inv-7 mutant, wild-type and ove

r-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 ANO

VA test.