Brassinosteroid signaling affects secondary cell wall deposition in cotton fibers

Industrial Crops and Products 65 (2015) 334–342

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Brassinosteroid signaling affects secondary cell wall deposition in cotton fibers Yan Sun a , Suresh Veerabomma a , Mohamed Fokar b , Noureddine Abidi c , Eric Hequet c , Paxton Payton d , Randy D. Allen b,∗ a

Department of Biological Sciences Texas Tech University, Lubbock, TX 79409, USA Institute for Agricultural Biosciences, Oklahoma State University, Ardmore, OK 73401, USA c Fiber and Biopolymer Research Institute, Texas Tech University, Lubbock, TX 79409, USA d USDA/ARS Cropping Systems Research Laboratory, Lubbock, TX 79415, USA b

a r t i c l e

i n f o

Article history: Received 6 June 2014 Received in revised form 11 November 2014 Accepted 13 November 2014 Available online 26 November 2014 Keywords: Brassinosteroid BRI1 Cellulose synthase Cotton fiber Gossypium hirsutum Secondary cell wall

a b s t r a c t Differentiation of cotton fibers involves sequential cell elongation and secondary cell wall deposition as they develop from fiber initials to highly elongated and thickened trichomes on the seed integument. Phytohormones appear to play important regulatory roles in the development of these economically important cells. Previous pharmacological experiments indicate that brassinosteroid (BR), along with auxin and gibberellins, promote cotton fiber elongation. To further evaluate the role of the BR signaling pathway in cotton fiber development, transgenic cotton plants that express transgenes designed to either over-express or suppress the expression of the BR receptor BRI1 were created. Analysis of the cotton fibers from these plants indicated that alteration of BRI1 expression had little effect on fiber length. However, over-expression of BRI1 led to increased fiber cellulose deposition while suppression of BRI1 expression strongly inhibited secondary cell wall development, resulting in fibers with reduced maturity. These alterations in fiber cell wall development corresponded with changes in cellulose synthase gene expression, indicating that secondary wall deposition is affected by BR-dependent modulation of cellulose synthase gene expression. These results indicate that BR signaling promotes the maturation of cotton fibers through the deposition of cellulose into the secondary cell wall. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Cotton fibers are highly elongated epidermal trichomes that grow from the seed integument of a small group of species in the genus Gossypium. Immediately after pollination, fibers undergo rapid polar elongation. Within about 18 days, they grow from initial cells, 20–30 ␮m in diameter, to hairs more than 2.5 cm in length (Delanghe, 1986). Subsequent deposition of large amounts of cellulose in the form of a thickened secondary cell wall gives the fiber its final shape and provides additional strength against breakage (Delmer and Amor, 1995). When dry, mature cotton fibers collapse and, if secondary wall thickness is optimum, take the shape of twisted ribbons, giving them the ability to intercalate with adjacent fibers when spun to form exceptionally strong yarn (see Aleman and Allen, 2010 for review).

∗ Corresponding author. Tel.: +1 580 224 0626; fax: +1 580 224 0624. E-mail address: [email protected] (R.D. Allen). http://dx.doi.org/10.1016/j.indcrop.2014.11.028 0926-6690/© 2014 Elsevier B.V. All rights reserved.

More than 90% of cotton fiber production in the Unites States is from Gossypium hirsutum (Upland cotton) with a smaller percentage of higher quality fiber coming from Gossypium barbadense (Pima or Sea Island cotton). The quality of cotton fibers depends primarily on their length, strength and fineness. Fineness is typically approximated by micronaire, although this measure is affected by both fiber diameter and fiber secondary wall thickness. A “fine” cotton fiber that is optimum for spinning into fine textiles has a small fiber perimeter that is well-filled, but not overly filled, with secondary wall cellulose. These primary quality characteristics are determined by genetic factors and the environmental conditions under which the crop is grown. Brassinosteroids (BR) are required for a wide range of plant developmental processes including responses to light, shoot and root elongation, seed development and germination, and development of vascular tissue (see Schumacher and Chory, 2000 for review). Arabidopsis plants that are homozygous for brassinosteroid insensitive 1 (bri1) exhibit many of the same characteristics as BR biosynthetic mutants such as det2 and cpd including reduced growth and apical dominance, delayed flowering and senescence,

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and male sterility (Clouse et al., 1993, 1996). BRI1 encodes a leucine-rich repeat receptor protein kinase (Li and Chory, 1997) that is expressed throughout Arabidopsis plants and localized in the plasma membrane (Friedrichsen et al., 2000). Over-expression of a BRI1-GFP fusion protein in Arabidopsis increased cell elongation and the number of BR binding sites in the plasma membrane (Wang et al., 2001) and BR was shown to bind directly to the extracellular domain of BRI1, indicating that it is a bona fide BR receptor (Kinoshita et al., 2005). The tetraploid cotton genome contains two functional BRI1 orthologs derived from the A and D sub-genomes (Sun et al., 2004). Both of these GhBRI1 genes were able to complement the bri1-5 allele when expressed in Arabidopsis. These genes are expressed at detectable levels in all cotton tissues tested but expression was elevated in rapidly expanding tissues such as hypocotyls and elongating cotton fibers. Brassinosteroids, along with auxins and gibberellin (GA), promote cotton fiber initiation and elongation in the cultured ovule system while the BR biosynthesis inhibitor Brassinazole2001 (Brz) strongly inhibits fiber elongation in cultured ovules (Sun et al., 2004, 2005; Shi et al., 2006). In addition, over-expression of a BR-regulated xyloglucan endotransglucosylase/hydrolase (XTH) in transgenic cotton plants resulted in the production of longer mature fibers (Allen et al., 2000; Lee et al., 2010) indicating that increased expression of this BR-responsive gene can promote fiber elongation. Based on these preliminary data, we hypothesized that BR signal transduction plays a role in determining the length of cotton fibers. To test this hypothesis, we created transgenic cotton plants with altered BRI1 expression. Surprisingly, mature fibers produced by these plants were similar in length to those of wildtype plants. However, fibers from plants that over-express BRI1 produced thicker secondary cell walls while antisense suppression of BRI1 expression reduced secondary cell wall deposition. These characteristics correspond with changes in cellulose synthase gene expression during fiber development. These results indicate that the BR signaling pathway plays an important role in regulating the maturation of cotton fibers in planta.

2. Materials and methods 2.1. Vector construction and cotton transformation Since BRI genes in both Arabidopsis and cotton do not have introns (Sun et al., 2004), the full length protein coding sequence (3591 bp) of AtBRI1 (AF017056) was amplified from Arabidopsis genomic DNA with primers that introduced BamHI sites to both 5 and 3 ends, and the amplified fragment was subcloned into the pGEM-T Easy Vector (Promega Inc., WI, USA). After confirming sequence integrity, the BRI1 coding sequence was subcloned into the dephosphorylated BamHI site of the intermediate vector pRTL3, placing it down stream of an enhanced CaMV 35S promoter. A 600 bp fragment from the 3 -end of the coding sequence of GhBRI1 was amplified and inserted into the same vector, in reverse orientation relative to the CaMV 35S promoter. These two gene constructs were then subcloned into the binary vector pCGN1578 that contains a p35S::NPTII selectable marker gene. The resulting binary plasmids were transformed into Agrobacterium tumefaciens strain EHA101. Hypocotyls from cotton seedlings (G. hirsutum cv. Coker 312, PI 529278) were used as explants for inoculation. Transformed calluses selected on tissue culture media containing kanamycin were transferred to liquid media for suspension culture, and then plated to induce somatic embryos. Plantlets that developed from those embryos were transferred to soil and grown to maturity in a greenhouse. PCR analysis was performed to confirm the insertion of the asBRI1 and oeBRI1 transgenes with a forward primer specific

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for the CaMV 35S promoter (5 -GCACAATCCCACTATCCTTCG-3 ), along with the reverse primers specific for GhBRI1 or AtBRI1 coding sequences (GhBRI1-A 5 -AAGAGGGCTGGCGTTTCTTC-3 ; AtBRI1-O 5 -TCATAATTTTCCTTCAGGAACT-3 ). 2.2. Fiber quality analysis Seed cotton was harvested by hand from mature first position bolls of the first 4 nodes of all plants tested and the fiber was manually removed from the seed to avoid mechanical breakage. Field-grown plants were cultivated during the 2007 growing season at the USDA Cropping Systems Laboratory in Lubbock, TX. Plants were grown in triplicate plots in a randomized block design. The field was equipped with a subsurface drip irrigation system adjusted to provide 70% evaporative transpiration replacement. Cotton samples were tested at the Texas Tech University Fiber and Biopolymer Research Institute on High Volume Instrument (HVI 900A, Uster, Knoxville, TN) for strength (10 determinations per sample), length (10 determinations per sample) and micronaire (4 determinations per sample). This system is the industry standard for evaluation of cotton fiber quality and all cotton produced and marketed in the U.S.A. is HVI tested. 2.3. Analysis of fiber structure For microscopic observation, representative mature dry fibers for each line were placed on slides and observed and photographed with a dissecting microscope using indirect lighting. Using this method, differences in diffraction caused by crystalline cellulose deposition in the thickened secondary cell walls are apparent. Fibers with thick secondary cell walls appear bright due to this diffraction while thinner walled fibers appear less bright. For structural analysis, cotton fiber samples were embedded in methacrylate polymer and sectioned with a MT 990 rotary microtome. The 1 ␮m thick sections were placed on a drop of water on albumin-coated slides and the slides were then placed on a glass rod support in a Petri dish above chloroform for 3 s to expand the sections. The slides were then placed on the hot plate to dry and fix the cross sections to the slide and then washed in methyl ethyl ketone to remove the methacrylate polymer leaving the fiber cross sections in place on the slide. The prepared slides were viewed with a microscope at 40× magnification and digital images were captured using a Hitachi 3 CCD Camera Model HVC-20 with a Coreco Oculus TCX Frame Grabber. The images were then measured for area and perimeter of both the fiber and lumen (Hequet et al., 2006). 2.4. Gene expression analysis Flowers were tagged on the day of anthesis and immature ovules were collected at 10 days post-anthesis (DPA), 15 DPA, 20 DPA, and 30 DPA. Fiber was removed from the ovule and samples were ground to powder in liquid nitrogen and total RNA was extracted using the modified hot borate method (Wan and Wilkins, 1994). Cotton RNA samples were quantified using a Nanodrop spectrophotometer (Nyxor Biotech). Quantitative real-time PCR was used to assay gene expression levels. Primer Express (Version 2.0) (ABI, CA, USA) was used to design primers and probes for real-time PCR analysis (Supplemental Table 1). Expression of the selected genes in different cotton cDNA samples were analyzed using TaqMan assays and 18S rRNA assays were used to normalize the data for RNA input. Cotton RNA samples were treated with DNase I (Promega) and diluted to 200 ng/␮l. Reverse transcription was carried out using random hexamers and TaqMan Reverse Transcription Reagent Kit (ABI) was used for first strand synthesis in 50 ␮l reactions (containing 1 ␮g of total RNA) at 37 ◦ C for 60 min. TaqMan PCR Core Reagents Kit (ABI)

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Fig. 1. Diagram of gene constructs used for development of transgenic cotton plants with altered BR responsiveness. For over expression of the BR receptor BRI1 (oeBRI1), the Arabidopsis BRI1 open reading frame sequence (AtBRI1 ORF) was inserted, in sense orientation, between an enhanced CaMV 35S promoter and nos terminator/poly A addition sequence. For suppression of BRI expression, a ∼600 bp fragment of the cotton BRI1 coding sequence (GhBRI1 ORF) was inserted in antisense orientation relative to the same promoter and terminator sequences.

was used for 50 ␮l PCR reactions as follows: 50 ◦ C for 2 min, 95 ◦ C for 10 min, and 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 60 s. Each sample was assayed three times. The relative expression levels of all the samples were calculated according to the ABI PRISM 7700 Sequence Detection System, User Bulletin #2 (Livak and Schmittgen, 2001). Western blots were used to assay the levels of BRI1 polypeptides in the positive transgenic plants. Cotton tissue samples were ground to powder in liquid nitrogen and proteins were extracted from about 200 mg tissues for each sample using the Plant Total Protein Extraction Kit (Sigma). Equal amounts of total protein from the asBRI1 and oeBRI1 transgenic and control plants, along with Arabidopsis leaf protein were loaded for electrophoresis in 10% Tris–HCl Ready Gels (Bio-Rad). After electrophoresis, proteins were electro-transferred onto PVDF immobilon-FL transfer membranes (Millipore). Non-specific binding sites were blocked by 5% non-fat milk for 1 h. After washing with TTBS (20 mM Tris, 500 mM NaCl, 0.05% Tween-20, pH 7.5), blots were incubated with polyclonal antibodies raised against AtBRI1 or cytosolic glyceraldehyde-3phosphate-dehydrogenase (GAPc) for 2 h. The blots were washed three times (10 min each) with TTBS, and incubated with the biotinylated goat anti-rabbit second antibody for 1 h, and washed again with TTBS. Streptavidin-linked alkaline phosphatase (BioRad) was added and the blots incubated for 1 h, then washed with TTBS. Finally, the BRI1 and GAPc were visualized using an alkaline phosphatase color development kit (Bio-Rad). Images of developed blots were quantified using a Kodak 1D Image Analysis Software (Eastman Kodak Company, NY, USA). The mass unit for intensity of each band was compared with the mass unit of the corresponding GAPc to obtain a relative value. 3. Results 3.1. Modification of BRI1 expression in transgenic cotton plants To modify the responsiveness of cotton plants to BR, transgenic cotton plants were developed that either over-express the BR receptor BRI1 or have reduced BRI1 expression due to antisense suppression. For BRI1 over-expression (oeBRI1), the full length BRI1 coding sequence from AtBRI1 was expressed under the control of a dual-enhancer CaMV 35S promoter (Fig. 1). For antisense suppression (asBRI1), a portion of the GhBRI1 coding sequence was inserted in antisense orientation relative to the same promoter. These gene constructs were transformed into A. tumefaciens and used to transform cotton tissue explants. Transformed calluses were selected and transgenic plants were regenerated via somatic embryogenesis. Regeneration of oeBRI1 plants to the somatic embryo stage proceeded rapidly and 15 independentlytransformed oeBRI1 transgenic cotton plants were successfully regenerated and these plants appeared to be normal with respect to vegetative growth and flowering. Six independently transformed osBRI1 T0 plants were selected, based on their expression levels and fertility, for further analyses.

Regeneration of asBRI1 plants proceeded more slowly than is typical and relatively few embryogenic cell lines were produced. After somatic embryogenesis, many of the regenerating plantlets failed to thrive. Ultimately, 11 independent asBRI1 plants were regenerated. Many of these regenerated asBRI1 plants were severely dwarfed and sterile (see Supplemental Fig. 1A) but some (6) of the asBRI1 plants grew more normally although the petioles and internodes of fruiting branches of the T0 plants were shortened relative to non-transgenic control plants resulting in a more compact growth habit (see Supplemental Fig. 1B). The phenotype of the dwarf asBRI1 plants suggested strong suppression of BRI1 expression resulting in BR insensitivity; however, since these plants were sterile and did not produce seed even when cross-pollinated with pollen from wild-type plants, they could not be used to evaluate the role of BRI1 in fiber development. However, the asBRI1 plants that developed more normally were fertile and produced sufficient seed for fiber analysis and for the propagation of T1 asBRI1 plants. The presence of the transgenes in the regenerated plants was confirmed by PCR analysis with primers specific for the CaMV 35S promoter, and AtBRI1 or GhBRI1 sequences. Preliminary RT-PCR analyses were used to identify several T0 transgenic plants with consistently high levels of transgene expression for further analyses. 3.2. Reduced sensitivity to BR in asBRI1 transgenic cotton plants Root growth is inhibited by exposure to exogenous brassinosteroid (Li et al., 2001). Therefore, the sensitivity of oeBRI1 and asBRI1 transgenic plants to brassinolide (BL) was analyzed using a root growth inhibition assay. The elongation of roots of non-transgenic Coker 312 (C312) seedlings was strongly inhibited by exposure to 1 ␮M BL (Fig. 2A). Although no significant differences in BLdependent root growth inhibition between the oeBRI1 and C312 seedlings were seen under these conditions, elongation of the tap roots of asBRI1 seedlings was less sensitive to BL-induced inhibition, resulting in a two-fold difference in root fresh weight between asBRI1 and C312 seedlings (Fig. 2B). Therefore, although the asBRI1 plants had nearly normal vegetative growth, they were detectably less sensitive to BR than wild-type plants. 3.3. Altered gene expression in fibers of BRI1 transgenic plants Levels of BRI1 mRNA in fibers of transgenic plants were assayed by quantitative real-time PCR (qPCR) using total RNA samples from fibers of transgenic and non-transgenic C312 plants at defined developmental stages. Fibers from oeBRI1 plants expressed high levels of AtBRI1 transcripts at 10 DPA. At this developmental stage, which correlates with rapid fiber elongation, transgene-derived AtBRI1 mRNA was increased by an average of 3-fold relative to endogenous GhBRI1 mRNA in oeBRI1 plants (Fig. 3A). Antisense GhBRI1 transcripts were strongly expressed in asBRI1 plants (see Supplemental Fig. 2) and expression of endogenous GhBRI1 mRNAs in fibers of asBRI1 plants was reduced relative to wild-type C312 plants by an average of 30% at 10 DPA and by more than 50% at 15 DPA (Fig. 3A). To determine if alterations in the expression of BRI1 transcripts in transgenic cotton plants correlated with changes in BRI1 protein accumulation, steady state levels of BRI1 was analyzed by Westernblot assays in leaves and 15 DPA fibers of C312 plants and selected oeBRI1 and asBRI1 transgenic plants. The blots were probed with anti-AtBRI1 polyclonal antibodies (Wang et al., 2001) and analyzed by densitometry. Levels of BRI1 in both leaves and fibers of oeBRI1 plants were 30–40% higher than in corresponding tissues of C312 plants (Fig. 3B). Levels of BRI1 in asBRI1 plants were reduced by 50% in leaves and by 70% in fibers, relative to C312 plants. While changes in BRI1 in asBRI plants correlate well with the reduction in BRI1

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Fig. 2. Root inhibition analysis of brassinolide (BL) sensitivity of asBRI1 cotton seedlings. (A) Coker 312 and three asBRI1 lines were used for root inhibition assay. Growth of both primary and secondary roots of Coker 312 seedlings was strongly inhibited by treatment with 1 ␮M BL, while tap roots of asBRI1 plants were less strongly inhibited, indicating reduced sensitivity to BL. (B) Root fresh weight was measured 10 days after germination. The values are means of measurements of 5 seedlings from three lines for each treatment with three repeats. Bars = mean ± SD, *significantly different from C312 (p ≤ 0.05).

transcripts, the comparatively small increases in BRI1 accumulation in oeBRI plants could indicate that BRI1 expression is subject to post-transcriptional regulation.

3.4. Altered BRI1 expression affects cotton fiber characteristics Mature cotton fibers were removed from T0 seeds harvested from greenhouse-grown transgenic oeBRI1, asBRI1 and C312 cotton plants for quality evaluation using HVI. Fiber length values reported by HVI represent the mean length by number of the longest 50% of the fibers by weight in the sample. The upper half mean length of fibers from oeBRI plants, which measured 29.0 mm, was somewhat less than those from non-transgenic C312 plants while no significant differences in fiber length were detected between fibers from asBRI1 transgenic and C312 plants, with upper half mean lengths of 32 mm and 31.5 mm respectively (Table 1). Micronaire is an indirect measurement of fiber surface area that is assayed by air flow passing through a plug of fibers. Since the flow of a fluid through a plug of particles is inversely proportional to the square of the specific surface (Kozeny, 1927), micronaire reflects the combined effects of fiber perimeter and maturity (secondary cell wall thickness relative to fiber diameter). HVI analysis showed

that fiber samples from oeBRI1 plants had micronaire values that were approximately 10% higher than fiber from non-transgenic C312 plants (Table 1). Conversely, the micronaire of samples from asBRI1 plants was reduced by about 20% relative to wild-type plants. The mean micronaire value of fiber samples from six independently transformed oeBRI1 plants was 5.1, compared to 4.6 for C312 plants, while fibers from six independently transformed asBRI1 plants had a mean micronaire value of 3.7. Similar results

Table 1 Comparison of cotton fiber characteristics using HVI measurements. Mature fibers from first position bolls of greenhouse-grown non-transgenic plants (Coker 312) and transgenic T0 BRI1 over-expressing (oeBRI1) or antisense suppressed (asBRI1) plants were tested. Fiber samples from six-independently-transformed lines for each transgene were evaluated. Data are means (±SD) of 10 measurements of each sample for length and strength and 4 measurements each for micronaire. Genotype

Coker 312 oeBRI1 asBRI1 *

HVI Length (mm)

Strength (kN m kg−1 )

Micronaire

31.5 ± 0.66 28.9 ± 0.78* 32.0 ± 0.76

294.0 ± 8.92 327.3 ± 20.29* 315.6 ± 9.90*

4.6 ± 0.30 5.1 ± 0.25* 3.7 ± 0.21*

Significantly different from Coker 312 (p ≤ 0.05).

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Y. Sun et al. / Industrial Crops and Products 65 (2015) 334–342 Table 2 HVI measurements of fiber characteristics from cotton plants grown under irrigated field conditions. Mature fibers were collected from first position bolls of field-grown Coker 312, and T3 homozygous expressing and non-expressing (null) segregant plants each derived from three-independently-transformed BRI1 over-expressing (oeBRI1) or antisense suppressed (asBRI1) transgenic lines. Plants were grown in three randomized plots. Data are means of 10 measurements of each sample for length and strength and 4 measurements each for micronaire. Overall means (±SD) for Coker 312/null lines, oeBRI1, and asBRI1 lines are shown. Genotype

HVI Length (mm)

Strength (kN m kg−1 )

Micronaire

C312/null Coker 312 oeBRI1-null asBRI1-null

31.8 ± 1.32 32.00 30.99 32.51

285.0 ± 19.50 281.9 271.5 301.5

4.7 ± 0.16 4.7 4.7 4.6

oeBRI1 oeBRI1-1 oeBRI1-2 oeBRI1-3

32.7 ± 1.08 33.02 33.00 32.26

269.5 ± 6.86 275.4 265.6 267.5

5.3 ± 0.07* 5.3 5.3 5.4

asBRI1 asBRI1-1 asBRI1-2 asBRI1-3

31.8 ± 1.56 33.53 32.00 29.72

284.9 ± 13.62 295.0 293.0 266.6

4.1 ± 0.13* 4.2 4.3 3.6

*

Significantly different from Coker 312 (p ≤ 0.05).

5.3, about 15% greater than the mean value for non-expressing plants (4.7). Fiber from asBRI1 plants had micronaire measurements of 4.1, about 10% lower than that from non-expressing plants (Table 2). Fig. 3. Analysis of expression of transgene-derived and native BRI1 in transgenic asBRI1 and oeBRI1 cotton plants. (A) Top left panel – Levels of AtBRI1 mRNA in fibers of oeBRI1 plants at 10 days post anthesis (DPA) were 3-fold higher than native GhBRI1 transcripts. Bottom left panel – Levels of native GhBRI1 transcripts in 15 DPA fibers from asBRI1 were 50% lower than in comparable C312 cotton fibers. Right panels – Levels of ubiquitin mRNAs in the same RNA samples. Bars = mean ± SD, *significant differences from C312 (p ≤ 0.05). (B) Western blot detection of BRI1 polypeptides in protein extracts from young leaves and fibers (15 DPA) of transgenic asBRI1 and oeBRI1 plants compared with non-transgenic plants (C312). Blots were probed with anti-AtBRI1 antibodies. Duplicate blots were probed with antibodies against glyceraldehyde-3-phosphate-dehydrogenase (GAPc) as loading controls. Relative densities are indicated above the bands.

were obtained from HVI analysis of fiber from greenhouse-grown T1 transgenic plants. Cotton fiber strength is determined with HVI by measuring the mass-normalized force required to break a fiber bundle and is recorded as kN m kg−1 . Although variation was seen in fiber strength measurements of fiber from oeBRI1, asBRI1 and C312 plants (Table 1), these differences were not consistent between generations or under different growth conditions (see Table 2). To test whether the differences in fiber quality characteristics seen in greenhouse-grown transgenic cotton plants with altered BRI1 expression were stable under typical agricultural conditions, homozygous T3 plants of three independent oeBRI1 and asBRI1 transgenic lines, along with non-transgenic C312 plants and null segregants selected from oeBRI1 and asBRI1 lines were grown in the field under irrigated conditions. Mature fiber samples from first position bolls of these plants were harvested and analyzed using HVI (Table 2). No significant differences were detected between non-transgenic C312 plants and non-expressing null segregants derived from either oeBRI1 or asBRI1 transgenic lines for any of the HVI fiber quality parameters. Consistent with data from fiber of greenhouse grown plants, no significant differences were detected for fiber length or strength between non-expressing plants and oeBRI1 or asBRI1 plants. However, under these conditions, the mean micronaire value for fiber from oeBRI1 plants was

3.5. Altered BRI1 expression affects cotton fiber secondary cell walls While fiber quality analyses indicated that alterations of BRI1 in transgenic cotton plants affected fiber micronaire, differences in fiber characteristics were readily visible between fibers of asBRI1, oeBRI1 and C312 plants using light microscopy. When viewed in this way, mature cotton fibers from greenhouse-grown C312 plants had a characteristic twisted-ribbon structure and a bright appearance that is due to their thickened secondary cell walls, which are composed primarily of highly refractive crystalline cellulose (Fig. 4A). Fibers from oeBRI1 plants were also twisted and appeared to be thicker and more refractive than those from non-transgenic plants, which is likely due to more extensive secondary cell walls. Conversely, fibers from asBRI1 plants were less twisted and were less refractive in appearance relative to C312 plants, probably indicating reduced amounts of cellulose in their secondary cell walls. Observation of fiber from bolls of C312 plants treated with Brz to inhibit BR biosynthesis (Sekimata et al., 2001) also showed flattened fibers without twists similar to those from asBRI1 plants (Fig. 4A). To directly assess effects of altered BRI1 expression on fiber cell wall deposition, fibers from greenhouse-grown oeBRI1, asBRI1, and C312 plants were embedded in a methacrylate polymer and sectioned and the thickness of the cell walls was measured from light micrographs. Fibers from C312 bolls treated with BL and Bzr were also analyzed. Clear differences were seen in cell wall thickness between fibers from seeds of C312, oeBRI1, and asBRI1 plants and from bolls of C312 plants treated with Bzr (Fig. 4B). Quantitative image analyses (Hequet et al., 2006) indicated that cell walls of fibers from oeBRI1 transgenic cotton lines were, on average, 15% thicker than fibers from C312 plants (Fig. 4C) while the average fiber cell wall thickness for asBRI1 plants was reduced by nearly 40% relative to wild-type plants. Brz-treated bolls from C312 plants produced fibers with reduced cell wall thickness similar to those of asBRI1 plants.

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Fig. 4. Comparison of secondary cell wall characteristics in mature fibers from transgenic oeBRI11, asBRI1 and non transgenic Coker 312 cotton plants. (A) Top panel – micrographs of fiber from representative plants show reduced secondary cell wall development in fibers from asBRI1 plants compared to those from Coker 312 plants as indicated by lower levels of refraction. Fiber from representative oeBRI1 plants showed increased refraction compared to Coker 312 fibers indicating more extensive secondary cell wall development. For comparison, fiber from intact Coker 312 bolls treated with the BR biosynthetic inhibitor Brz2001 (Brz) have very little secondary cell wall and showed very low refraction. Scale = 0.5 mm. Lower panel – representative images used for analyses of cotton fiber cross-sections showed clear differences in secondary cell wall deposition between transgenic plants and Coker 312 plants while cell walls of fiber from Brz-treated bolls were extremely thin. (B) Quantitative image analysis of fiber cross sections. More than 2000 fiber cross-sections were analyzed for each data point. Bars = mean ± SD, *significant differences from C312 (p ≤ 0.05).

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Fig. 5. Quantitative real-time PCR analysis of cellulose synthase gene expression in cotton plants. (A) Left panel – comparison of GhCesA1 transcript levels in developing fibers from asBRI1 and oeBRI1 transgenic plants and non-transgenic plants (C312) from 10 DPA through 30 DPA. (B) Left panel – comparison of GhCesA1 mRNA expression in fiber from intact C312 bolls treated with either brassinolide (BL), Brassinazole2001 (Brz) or a mock treatment (MK). Right panels – comparison of ubiquitin transcripts from the same total RNA samples. Points = mean ± SD, *significant differences from C312 (p ≤ 0.05).

3.6. BR regulates cellulose synthase gene expression Since the secondary cell walls of cotton fibers are composed of >90% cellulose, the differences in cell wall thickness between fibers from oeBRI1, asBRI1 and control plants are likely to correlate with differences the cellulose biosynthesis. Therefore, quantitative realtime PCR was used to measure the relative transcript levels of two cellulose synthase genes, GhCesA1 and GhCesA2, which are highly expressed in cotton fibers during secondary cell wall deposition (Pear et al., 1996). Total RNA samples were extracted from fibers of oeBRI1, asBRI1, and C312 plants at different developmental stages. RNA samples from cotton fibers of BL-, Brz-, and mock-treated bolls of C312 plants were also prepared. Since the expression patterns of GhCesA1 and GhCesA2 were nearly identical, only data for GhCesA1 are shown in Fig. 5. Expression of mRNAs from both cellulose synthase genes was approximately 2-fold higher in oeBRI1 fibers than in fibers from C312 plants at 15 and 20 DPA (Fig. 5A). Likewise, expression of these transcripts in fibers of BL-treated C312 bolls was also substantially increased (Fig. 5B). Conversely, expression of GhCesA1 and GhCesA2 mRNAs in fibers from asBRI1 plants was reduced by nearly 70% compared to fibers from C312 plants at 15 DPA and expression of these genes in fibers from Brz-treated bolls of C312 plants was also greatly reduced (Fig. 5B). For comparison, expression of a cotton ubiquitin transcript (AY189972) exhibited a different developmental pattern and expression of this gene did not respond to alterations in BRI1 expression in transgenic plants or to BL or Bzr treatments. These results indicate that expression of secondary cell wall-specific cellulose synthase genes in cotton responds to BR signaling. 4. Discussion Based primarily on experiments with cultured cotton ovules, it is known that exogenously supplied GA and auxin promote cotton fiber initiation and elongation (Kosmidou-Dimitropoulou, 1976; Dhindsa, 1978; Sharma et al., 1995). Application of GA and IAA to developing flowers of intact plant was also shown to increase the rates of fiber initiation (Seagull et al., 2000; Seagull and Giavalis,

2004). On the other hand, application of abscisic acid inhibited fiber elongation in cultured ovules (Beasley and Ting, 1974; Nayyar et al., 1989) and an inverse correlation between ABA content and elongation in developing fibers was reported (Chen et al., 1996; Gokani et al., 1998). Cytokinins also inhibit fiber development in ovule cultures (Beasley and Ting, 1973) and high levels of cytokinin were detected in a short fiber mutant of cotton (Chen et al., 1997). Although ethylene was thought to be an inhibitor of fiber growth, Shi et al. (2006) showed that exogenous ethylene promoted robust fiber expansion in cultured ovules and inhibition of ethylene synthesis suppressed fiber growth. Addition of BR, along with auxin and GA to ovule cultures, synergistically promoted fiber elongation, while addition of Brz inhibited fiber elongation (Sun et al., 2005; Shi et al., 2006). Antisense suppression of the BR biosynthetic gene GhDET2 in cotton reduced regeneration rates and viable antisense GhDET2 transgenic plants were stunted and aborted their fruit within 3–5 DPA (Luo et al., 2007). However, scanning electron microscopic analysis of ovules at 0–1 DPA showed that fiber initiation was inhibited and fiber elongation could be restored on these ovules in culture by application of exogenous BR. While transgenic cotton plants that constitutively over-expressed GhDET2 were also sterile, plants that expressed GhDET2 under control of a seed coat-specific promoter developed normally and showed an increase in fiber number and the length of the longest fibers (measured manually with a ruler). Analysis of secondary cell wall development of fibers from these plants was not reported. Based on these results, we predicted that transgenic enhancement of BR responsiveness in cotton plants would promote cell elongation and result in longer fibers, while suppression of BRI1 expression was expected to inhibit cell elongation. However, our results show that alteration of BRI1 expression in transgenic plants appears to have little influence on average fiber length but strongly affects secondary cell wall deposition. Over-expression of BRI1 in transgenic cotton plants resulted in fibers with thicker cell walls while fibers from transgenic asBRI1 cotton plants showed reduced secondary cell wall development (Fig. 4). Inhibition of BR biosynthesis in planta by treatment of developing bolls with Brz produced fibers with similar characteristics (Fig. 4). These changes in fiber structure correlate with altered expression of cellulose synthase genes involved in secondary cell wall deposition (Pear et al., 1996) (Fig. 5), indicating that secondary cell wall development in cotton fibers is controlled, at least in part, through the direct or indirect modulation of cellulose synthase gene expression by the BR signaling pathway. Although the effects of phytohormones on the initiation and elongation of cotton fibers are well characterized, their role in secondary wall formation is not as well understood. Kim and Triplett (2005) reported that both ABA and IAA up-regulated the expression of secondary cell wall-specific cellulose synthase genes GhCesA1 and GhCesA2 in cultured cotton ovules while expression was suppressed by GA. Our results show that BR signaling is also critical for secondary cell wall development, indicating that regulatory crosstalk between the ABA, auxin, and BR signaling pathways may be involved in the regulation of cotton fiber maturation. ABA, auxin, and BR regulate many genes independently (Zurek and Clouse, 1994; Goda et al., 2004) but linkage between the signaling pathways for these phytohormones is apparent in many plant systems. For example, auxin and BR act synergistically to control hypocotyl elongation in a number of species (Mandava, 1998; Goda et al., 2002; Nemhauser et al., 2004). While ABA and BR have antagonistic effects on plant growth and seed germination, both can induce stress-responsive gene expression, leading to increased abiotic stress tolerance (Kagale et al., 2007; Bajguz and Hayat, 2009). Transcriptional profiling results indicate overlap between ABA, auxin and BR signaling pathways at the level of

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transcriptional regulation (Nemhauser et al., 2004). IAA5, IAA19 and SAUR-AC1 are induced by both BR and auxin (Nakamura et al., 2003) and iaa7/axr2 and iaa17/axr3 mutants show aberrant BR responses (Nakamura et al., 2006). Furthermore, the BR-regulated GSK3-like kinase BIN2 phosphorylates ARF2 and limits its repression of auxinresponsive gene expression (Vert et al., 2008). Analysis of the 5 flanking sequences of genes that respond to both BR and auxin showed enrichment of a putative auxin/BR responsive cis-acting element known as ARFAT (Nemhauser et al., 2004; Nakamura et al., 2006). The 5 flanking sequences of GhCesA1 and GhCesA2 genes both contain canonical ARFAT elements, along with putative Ebox sequences, identified as BR responsive cis-acting elements in several Arabidopsis genes (Yin et al., 2005). Thus, expression of cellulose synthase genes in cotton may be regulated by complex hormonal interactions. Low fiber maturity is a significant obstacle for the cotton industry. This problem is exacerbated in areas with short growing seasons and the adoption of high-yielding varieties with longer maturation time requirements. It is possible that genetic modification of BRI1 expression could be used to accelerate fiber maturation in these varieties. Moreover, alteration of downstream components of the BR signal transduction pathway may provide additional targets for genetic modification of cotton fiber development. Acknowledgements The authors wish to acknowledge the generous gift of Brz2001 from Dr. Tadao Asami, and Dr. Shigeo Yoshida, RIKEN, Japan. We also thank Dr. Joanne Chory, Salk Institute for Biological Studies, La Jolla, CA for the anti AtBRI1 antibody. This project was supported by USDA NRI CSREES grant number 2003-35304-13384 and Oklahoma Agricultural Experiment Station project number OKL02714. DNA sequencing was performed at the Texas Tech University Center for Biotechnology and Genomics. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop. 2014.11.028. References Allen, R.D., Burns, T.M., Light, G., Fokar, M., 2000. Investigating the role of xyloglucan endotransglycosylase in cotton fiber quality. In: Benedict, C., Jividen, G. (Eds.), Genetic Control of Cotton Fiber and Seed Quality. Cotton Inc., Cary, NC, pp. 166–174. Aleman, L., Allen, R.D., 2010. Research in cotton fibre development. In: Singh, B. (Ed.), Industrial Crops and Uses. CABI, Oxfordshire, UK, pp. 277–307. Bajguz, A., Hayat, S., 2009. Effects of brassinosteroids on the plant responses to environmental stresses. Plant Physiol. Biochem. 47, 1–8. Beasley, C.A., Ting, I.P., 1973. The effects of plant growth substances on in vitro fiber development from fertilized cotton ovules. Am. J. Bot. 60, 130–139. Beasley, C.A., Ting, I.P., 1974. Effects of plant growth substances on in vitro fiber development from unfertilized cotton ovules. Am. J. Bot. 61, 188–194. Chen, Y.N., Du, X.M., Zhou, X., Zhou, H.Y., 1996. Fluctuation in levels of endogenous plant hormones in the ovules of normal and mutant cotton during flowing and their relation to fiber development. J. Plant Growth Regul. 15, 173–177. Chen, Y.N., Du, X.M., Zhou, X., Zhou, H.Y., 1997. Levels of cytokinins in the ovules of cotton mutants with altered fiber development. J. Plant Growth Regul. 16, 181–185. Clouse, S.D., Hall, A.F., Langford, M., McMorris, T.C., Baker, M.E., 1993. Physiological and molecular effects of brassinosteroids on Arabidopsis thaliana. J. Plant Growth Regul. 12, 61–66. Clouse, S.D., Langford, M., McMorris, T.C., 1996. A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth development. Plant Physiol. 111, 671–678. Delanghe, E.S., 1986. Lint development. In: Mauney, J.R., Stewart, J.M.D. (Eds.), Cotton Physiology. The Cotton Foundation, Memphis, TN, USA, pp. 325–349. Delmer, D.P., Amor, Y., 1995. Cellulose biosynthesis. Plant Cell 7, 987–1000. Dhindsa, R.S., 1978. Hormonal regulation of ovule and fiber growth. Effects of bromodeoxyuridine, AMO-1618, and p-chlorophenoxyisobutyric acid. Planta 141, 269–273.

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