Supplementary: Supplementary Table 1 Source of the ... · PDF filetransformation method. ... Developmental control of H+/amino acid permease gene ... Samples for fiber quality and

Supplementary: Supplementary Table 1 Source of the promoters and their expression patterns in cotton. Promoter sequence was fused with the GUS or GFP marker gene, and the gene cassettes were delivered into cotton genome by Agrobacterium mediated transformation method. Their expression patterns were determined by GUS staining or GFP observation.

Promoters Source Expression pattern in cotton

SCFP1 Cotton 0~25 DPA fiber

E62 Cotton 5~24 DPA fiber

GhAlp* Cotton Root, hypocotyl, anther, ovule, seed and fiber

(>20 DPA)

GhSS33 Cotton Vascular tissue, stamen, pistil, pollen, ovary

and fiber (1~40 DPA)

GhCesA44 Cotton Fiber (secondary wall thickening stage, 5~35

DPA)

D1135 Cotton Seed (>20 DPA)

GhHOX2-1* Cotton Root, hypocotyl, receptacles, ovary and fiber

(0~12 DPA)

GhHOX2-2* Cotton Hypocotyl, stem, leaf, petal, ovary and fiber

(0~12 DPA)

FBP76, 7 Petunia Epidermal cell of ovule and fiber (-2~10

DPA)

BAN8 Arabidopsis Xylem of root, stem and petiole, epidermal

cell of ovule and fiber (-1~10 DPA)

AtAAP19 Arabidopsis Embryo (>25 DPA)

AtAAP29 Arabidopsis Endosperm (>20 DPA)

Agl510 Arabidopsis Stigma, receptacle, pollen, ovule and seed (-2

~30 DPA)

NtSC11 Tobacco Seed (>35 DPA)

Lefsm112 Tomato Ovary (-3~10 DPA) and pollen

PV13 Bean Seed (>20 DPA)

*Unpublished data Source of promoters: 1. Hou, L. et al. SCFP, a novel fiber-specific promoter in cotton Chin. Sci. Bull.

53, 2639-2645 (2008). 2. John, M.E. & Crow, L.J. Gene expression in cotton (Gossypium hirsutum L.)

fiber: Cloning of the mRNAs. Proc. Natl. Acad. Sci. 89, 5769-5773 (1992). 3. Luo, X. et al. Cloning and analysis of the upstream regulated sequence of a

cotton fiber sucrose synthase gene. Chinese Journal of Biochemistry and Molecular Biology 21, 335-340 (2005).

4. Kurek, I., Kawagoe, Y., Jacob-Wilk, D., Doblin, M. & Delmer, D. Dimerization of cotton fiber cellulose synthase catalytic subunits occurs via

Nature Biotechnology: doi:10.1038/nbt.1843

oxidation of the zinc-binding domains. Proc. Natl. Acad. Sci. 99, 11109-11114 (2002).

5. Luo, K., Guo, Y., Xiao, Y., Hou, L. & Pei, Y. Cloning and characterization of D-113 gene promoter from cotton. Acta Genetica Sinica 29, 161-165 (2002).

6. Colombo, L. et al. Downregulation of ovule-specific MADS box genes from petunia results in maternally controlled defects in seed development. Plant Cell 9, 703-715 (1997).

7. Luo, M. et al. GhDET2, a steroid 5α-reductase, plays an important role in cotton fiber cell initiation and elongation. The Plant Journal 51, 419-430 (2007).

8. Debeaujon, I. et al. Proanthocyanidin-accumulating cells in Arabidopsis testa: Regulation of differentiation and role in seed development. Plant Cell 15, 2514-2531 (2003).

9. Hirner, B., Fischer, W.N., Rentsch, D., Kwart, M. & Frommer, W.B. Developmental control of H+/amino acid permease gene expression during seed development of Arabidopsis. The Plant Journal 14, 535-544 (1998).

10. Savidge, B., Rounsley, S.D. & Yanofsky, M.F. Temporal relationship between the transcription of two Arabidopsis MADS box genes and the floral organ identity genes. Plant Cell 7, 721-733 (1995).

11. Pierre R. Fobert, H.L.J.C.S.G.-M.T.O.J.H.G.S.V.N.I.B.L.M. T-DNA tagging of a seed coat-specific cryptic promoter in tobacco. The Plant Journal 6, 567-577 (1994).

12. Barg, R. et al. The tomato early fruit specific gene Lefsm1 defines a novel class of plant-specific SANT/MYB domain proteins. Planta 221, 197-211 (2005).

13. Goossens, A., Dillen, W., De Clercq, J., Van Montagu, M. & Angenon, G. The arcelin-5 gene of Phaseolus vulgaris directs high seed-specific expression in transgenic Phaseolus acutifolius and Arabidopsis plants. Plant Physiol. 120, 1095-1104 (1999).


Supplementary Table 2 Phenotypes of transgenic cotton plants containing iaaM driven by different promoters

Promoter

Name Active in

Active

period

(DPA)

Promoter

activity

Number of

transformants

obtained

Number of

abnormal

plants*

Number of

normal

plants

E6 fiber 5~24 weak 13 1 12

SCFP fiber 0~25 strong 17 15 2

Lefsm1 ovule -3~10 strong 11 11 0

Agl5 ovule -2~30 strong 22 21 1

FBP7 seed coat -2~10 weak 23 1 22

*Mostly showed male-sterility or boll abortion


Supplementary Table 3 (a) Analysis of variance for the three-year field trials (2007~2009). Plants were grown in fields in Southwest University, Chongqing, China, by a randomized complete block design with three replications.

Source Degree of

freedom

Sum of

squares

Mean

squares F value

Genotype 2 443.26 221.63 209.09*

Year 2 1.09 0.55 0.50ns

Genotype×Year 4 4.24 1.06 0.97ns Lint%

Error 12 13.17 1.10

Genotype 2 0.17 0.08 2.50ns

Year 2 2.44 1.22 16.57*

Genotype×Year 4 0.13 0.03 0.46ns Seed cotton

Error 12 0.88 0.07

Genotype 2 0.65 0.33 222.03*

Year 2 0.52 0.26 14.52*

Genotype×Year 4 0.01 0.00 0.08ns Lint yield

Error 12 0.22 0.02

Genotype 2 3.89 1.95 70.97*

Year 2 0.05 0.03 1.66ns

Genotype×Year 4 0.11 0.03 1.72ns Micronaire

Error 12 0.19 0.02 * and ns, significant at 0.01 level and non-significant respectively.


Supplementary Table 3(b) Combined analysis of variance (ANOVA) for the two-year trial (2007-2008). Plants were grown in fields at Southwest University Chongqing, China by a randomized complete block design with three replications.

Source of

variation

Degree of

freedom

Sum of

squares Mean squares F value

Genotype 3 287.11 95.70 723.95*

Year 1 0.73 0.73 0.62ns

Genotype×Year 3 0.40 0.13 0.11ns Lint %

Error 12 14.08 1.17

Genotype 3 0.14 0.05 3.45ns

Year 1 1.70 1.70 14.61*

Genotype×Year 3 0.04 0.01 0.12ns Seed cotton

Error 12 1.39 0.12

Genotype 3 0.48 0.16 95.17*

Year 1 0.39 0.39 14.25*

Genotype×Year 3 0.01 0.00 0.06ns Lint yield

Error 12 0.33 0.03

Genotype 3 3.33 1.11 264.57*

Year 1 0.02 0.02 1.39ns

Genotype×Year 3 0.01 0.00 0.25ns Micronaire

Error 12 0.20 0.02 * and ns, significant at 0.01 level and non-significant respectively.


Supplementary Table 3 (c) Analysis of variance (ANOVA) for lint percentage and micronaire in cotton samples from two field trials (2007-2008) at Southwest University, Chongqing, China.

Year/Traits Source of variation Degree of

freedom

Mean

squares F value F valuez

Genotype 3 145.42 49.73***

Repeat × Genotype 8 2.92 41.64***

Time 2 14.59 207.79*** 207.79***

Genotype × Time 6 0.316 4.49** 4.49ns

2007 Lint

percentage

Error 16 0.07

Genotype 3 1.50 39.37***


Time 2 0.250 56.92*** 56.92***

Genotype × Time 6 0.003 0.73ns 0.73 ns

2007 Micronaire

Error 16 0.004

Genotype 3 235.88 25.61**


Time 4 20.82 136.14*** 272.29***

Genotype × Time 12 0.369 2.41* 4.82*

2008 Lint

percentage

Error 32 0.153

Genotype 3 3.312 38.28***

Repeat × Genotype 8 0.086 4.81**

Time 4 2.755 153.15*** 306.11***

Genotype × Time 12 0.009 0.54ns 1.08ns

2008 Micronaire

Error 32 0.018 z, F-tests using conservative degree of freedom, which require larger F-value to declare significant for time and genotype × time interaction. *, **, *** and ns, significant at 0.05, 0.01, 0.001 level and non-significant respectively. No interaction between sampling time and genotype reaches significant level, except the lint percentage in 2008.


Supplementary Table 4 Comparison of yield components of the FBP7::iaaM transgenic cotton and the control in 2008 field trial.

Line Bolls per plant Seeds per boll Lint per 100

seeds (g)

Weight per 100

seeds (g)

FM9 20.9a 24.1±0.3 9.8a 9.9a

FM14 20.5ab 24.4±0.2 9.3ab 10.2a

FM20 19.8bc 24.3±0.4 9.0ab 11.8b

Control 19.4c 24.2±0.3 8.6b 11.8b

Each data represents the mean ± S.D. (n=3). Control, segregated non-transgenic plants derived from FBP7::iaaM cotton. The significance of the difference was analyzed by Tukey’s multiple comparison test. Means followed by the same letter are not significantly different (P<0.05). The boll harvested after 10th October was not included in the analysis.


Supplementary Table 5 Comparison of fiber length and strength of the FBP7::iaaM transgenic cotton and controls in the 2007, 2008 and 2009 field trials.

Year Line Fiber length (mm) Fiber strength

(CN/tex)

FM9 30.4±0.2 30.1±0.4

FM14 30.1±0.5 30.7±0.4

FM20 30.4±0.7 30.0±0.2 2007

Control 30.4±0.3 30.1±0.6

FM9 31.1±0.6 29.8±0.7

FM14 30.8±0.5 29.5±1.1

FM20 31.4±0.3 30.2±0.8

Control 31.3±0.5 29.6±0.9

2008

Wild-type 30.7±0.3 29.7±0.3

FM9 29.5±0.4 30.9±0.6

FM14 29.2±0.4 30.4±0.2 2009

Control 30.1±1.0 31.2±1.6

Each data represents the mean ± S.D. (n=3). All the values in the table are not significantly different. Control, segregated non-transgenic plants derived from FBP7::iaaM cotton.


Supplementary Table 6 Cotton fiber yield and quality of FBP7::iaaM transgenic lines and control in the 2010 field trial.

Line

Seed cotton

yield

(kg/plot)

Lint yield

(kg/plot)

Lint

percentage

(%)

MicronaireFiber length

(mm)

Fiber

strength

(CN/tex)

FM9 30.31a 14.46a 47.8a 4.6a 29.7a 30.5a

FM14 28.20b 12.62b 44.8b 4.6a 28.8b 29.5b

Control 28.86b 10.74c 37.3c 5.3b 29.7a 30.5a

Plants were grown at Southwest University (Chongqing, China) in a randomized complete block design with three replications. The plot size was 100 m2 and the plant density was the same as that in 2007~2009. Samples for fiber quality and lint percentage were harvested from 11th Sep to 20th Sep, 2010 Standard deviations represent three measurements. The result was analyzed by Tukey’s multiple comparison test. Control, segregated non-transgenic plants derived from FBP7::iaaM cotton. Means followed by the same letter are not significantly different (P<0.05).


Supplementary Figure 1 Diagram showing the anatomy of the square, flower, boll and ovule of cotton.


Supplementary Figure 2 Effect of IAA and IAA inhibitor NPA on cotton fiber development in ovule culture. The -1-DPA ovules at the middle position of bolls were incubated for a week in BT medium (Beasley, C.A. 1973, Science 179, 1003-1005) containing 0.5 µM GA3 (Sigma, USA) and plus with: A. 5 µM IAA, B. 5 µM IAA + 50 µM NPA, C. 25 µM IAA, D. 25 µM IAA + 50 µM NPA. Samples were observed by an MVX-10 microscope (Olympus, Japan). Scale bars represent 1 mm.


Supplementary Figure 3 Schematic representation of expression cassettes for iaaM driven by different promoters. Each cassette was inserted into a modified binary vector pBI121-M for cotton transformation.


Supplementary Figure 4 Expression levels of iaaM in transgenic plants (T0) driven by FBP7 promoter. RNA from 0-DPA ovules was extracted for first strand cDNA synthesis. Expression levels of HISTONE3 (AF024716, GhHis) were used as the internal control. (a) RT-PCR analysis of the expression of iaaM gene in transgenic plants. (b) Real-time PCR analysis of the expression of iaaM. Each bar represents three repetitions from each RNA sample. WT, wild-type; P, the plasmid containing iaaM.


Supplementary Figure 5 IAA content in 0-DPA ovules of transgenic cotton line FM9, E6::iaaM23 and wild-type on the day of anthesis. The ovule samples were ground into powder and IAA was extracted in cold 80% (v/v) methanol. The extracted IAA was purified by a Sep-Pak® Plus tC18 cartridge (Waters, Ireland) and the concentration was determined by LC/MS (Finnigan, USA). Bars represent the standard deviation of three samples.


Supplementary Figure 6 Increased fiber initial density in cotton ovule resulting from exogenous application of IAA on the pedicels of -3-DPA buds. 10 µM IAA dissolved in lanolin (equal volume of water mixed with lanolin as control) was applied on the pedicels of -3-DPA squares. The 0-DPA ovules locating in the middle of a boll were fixed in 2.5% glutaraldehyde. Sample preparation followed the method of Sun et al. (2005, Plant Cell Physiol. 46, 1384-1391). Ovules were pictured by an S-3000N SEM (Hitachi, Japan). From the similar region of each SEM images, an area of 250×250 µm2 was selected for fiber initial counting. Cotton plants were grown in the greenhouse in the winter of 2008. The result was based on the data of nine ovules from three bolls. Bars represent standard deviation. Asterisks represent significant differences between transgenic lines and wild-type determined by t-test (*, P<0.05).


Supplementary Figure 7 Expression of FBP7::iaaL transgene resulted reduced IAA content in cotton ovules, which leads to a decrease in fiber initials. (a) Relative expression levels of iaaL gene in 2-DPA ovules of transgenic plants. Bars represent standard deviation based on three measurements from each RNA sample. (b) IAA content in the 0-DPA ovule of FBP7::iaaL transgenic cotton and the wild-type. Bars represent the standard deviation of three samples. (c) Comparison of fiber initial densities on ovules from FBP7::iaaL transgenic cotton (T1) and the wild-type. The 0-DPA ovules were pictured by a Hitachi S-3000N SEM. From the similar region of each SEM images, an area of 250×250 µm2 was selected for fiber initial counting. Cotton plants were grown in the greenhouse in the winter of 2009. The result was based on the data of nine ovules from three bolls. FL, FBP7::iaaL; WT, wild-type. Asterisks represent significant differences between transgenic lines and wild-type as determined by t-test (**, P<0.01).


Supplementary Figure 8 Fineness of fibers of transgenic cotton line FM9 and wild-type. (a) Cross-section of mature fibers from line FM9 (right) and the wild-type (left). Mature fibers were sectioned through the paraffin embedded section by 10 μm. Scale bars represent 50 μm. (b) Comparison of perimeters of fibers in transgenic cotton and the wild-type. A total of 300 individual transverse sections of fibers were photographed by a BX41TF microscope (Olympus, Japan) and were measured by software Image-Pro®Plus. Bars represent standard deviation. Asterisks represent significant differences between transgenic lines and wild-type, as determined by t-test (**, P<0.01).


Supplementary Figure 9 (a) Ginned seeds of transgenic cotton line FM9 and wild-type, showing the different amount of fuzzy fiber on seeds. Scale bar represents 1 cm. (b) Comparison of fuzz fiber contents between transgenic and wild-type cotton seeds. Acid-delinting method was used to remove fuzz fiber from ginned seeds. Fuzz content (%) = (weight of seeds - weight of delinted seeds) / weight of seeds × 100. FM, FBP7::iaaM; WT, wild-type. Bars represent standard deviations of three replicated measurements. Each sample was represented by100 ginned cotton seeds.


Supplementary Figure 10 (a)Histochemical localization of GUS activity in BAN::GUS transgenic cotton. Ovules at different developmental times (A, 0 DPA; B, 2 DPA; C, 5 DPA; D, 10 DPA; E, 1 DPA) were harvested and stained. The ovule image of 1 DPA (E) was represented by a paraffin embedded section (fi, fiber; oi, outer integument; ii, inner integument). Scale bars represent 1 mm (A~D) and 50 μm (E). (b) Lint percentage; (c) micronaire of BAN::iaaM transgenic cottons and wild-type. Seed cotton was harvested in October, 2010. Fiber quality was measured by the Cotton Fiber Quality Inspection and Testing Center, Ministry of Agriculture of China (Anyang city, Henan province, China). WT, wild-type; BM, BAN::iaaM. Bars represent the standard deviation of three measurements.


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Supplementary: Supplementary Table 1 Source of the ... · PDF filetransformation method. ... Developmental control of H+/amino acid permease gene ... Samples for fiber quality and