Introduction

Cotton (Gossypium hirsutum L.) is the dominant source of natural textile fiber and a significant oil crop, and has been a valued agricultural commodity for more than 8,000 years (Zhang et al. 2011). Cotton production is an important component of the global economy, affecting not only farmers in many countries but also consumers the world over (Lee et al. 2007). Many cotton improvement programs to enhance this crop have had the key goals of improving the yield and fiber quality (Shen et al. 2005). Cotton fiber is not part of the vascular tissue, and the appropriate botanical term for cotton fiber is trichome, which develops from the ovule epidermis (Kim and Triplett 2001). While the majority of plant trichomes are multicellular, the cotton trichome is unicellular and, apart from its economic importance, cotton fiber provides an excellent single-celled model for studying fundamental plant biological processes (Arpat et al. 2004; Kim and Triplett 2001). Cotton fiber length affects yarn strength, evenness, and spinning efficiency (Moore 1996; De Keyser et al. 2009). As a cotton fiber cell is highly elongated, its length basically represents its size and the trichome cell size affects yield as well as quality. The average length of cotton fibers varies with genotype and appears to be under strict control (Arpat et al. 2004; Fantes 1977; Ruan et al. 2009), about which little is known. Thus, information that can shed light on this control would be useful for developing strategies for improving cotton fiber quality and yield, features which are exceedingly difficult to incorporate into a single breeding program.

FCA, which encodes a strong promoter of the transition to flowering in Arabidopsis thaliana, contains two RNA recognition motif (RRM) domains and a WW protein interaction domain (Macknight et al. 1997). It has been previously found that the cell size and yield of rice (Oryza sativa) can be increased by ectopic expression of the first RRM domain of OsFCA (Hong et al. 2007). The second such domain of OsFCA can also increase cell size (Attia et al. 2005), suggesting that these OsFCA-RRMs each play a role in cell-size regulation. Designated here as Oryza sativa cell-size RRM 1 and 2 (Os-csRRM1 and Os-csRRM2, respectively), they exhibit a high degree of evolutionary conservation in plants. For Os-csRRM2 in particular, significant homology has been observed in Triticum aestivum, Hordeum vulgare, Lolium perenne, Zea mays, Ricinus communis, Vitis vinifera, Arabidopsis thaliana, and Brassica napus (90, 90 82, 81, 76, 68, 68, and 64% identity, respectively; Online Resource 1). This conservation suggests that the RRM domain might have similar functions in different plants; we have observed that overexpression of Bn-csRRM2 also increases cell size in B. napus (unpublished). As cotton fiber length is a key factor in cotton yield and quality, the possible enhancement of this attribute was investigated in the present study through the constitutive expression of Bn-csRRM2 in cotton plants.

Materials and methods

Cloning of target genes

Total RNA was extracted from Brassica napus using Trizol reagent (TianGen, Beijing, China). First-strand cDNA was synthesized with a PrimeScript RT reagent Kit (TaKaRa Bio. Co., Shiga, Japan) and used for polymerase chain reaction (PCR) amplification. Two primers were designed based on the Brassica napus FCA sequence (GeneBank accession no. AF414188): 5′-GAGGATCCATGGGTGCGGTAGAGTT-3′ (forward) and 5′-CGTAGATCTTGTGCCACTTCCCTTG-3′ (reverse) with restriction sites BamHI and XbaI (underlined), respectively. PCR cycling parameters were 94°C for 5 min, 30 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, and finally 72°C for 10 min. The products were cloned into pGEM-T Vector (Promega, Madison, WI, USA), recombinant plasmid pGEM-T-Bn-csRRM2 was produced, and then sequenced for verification.

Plasmid construction

The binary vector, pBin438 (derived from pBI121) was used as the basic vector (Li et al. 1994). It carries the kanamycin resistance gene for bacterial selection and the hygromycin phosphotransferase gene for plant transformation selection. It contains a 35S-35S promoter (a variant of the cauliflower mosaic virus 35S promoter with higher transcriptional activity) to drive transgene expression (Kay et al. 1987). The Bn-csRRM2 fragment was digested with BamHI/XbaI from plasmid pGEM-T-Bn-csRRM2 and ligated into the corresponding sites of pBin438 to result in the binary vectors pBin438-Bn-csRRM2 used for cotton transformation.

Transformation and regeneration of transgenic cotton plants

Bn-csRRM2 was introduced into cotton tissue by particle bombardment (Finer and McMullen 1990; Altpeter et al. 2005). In brief, surface-sterilized G. hirsutum cv. CCRI 12 seeds were husked and germinated in Murashige and Skoog (MS) medium. After 2–3 days, the meristems of hypocotyls were exposed by microscopic dissection, incubated in MS medium at 15°C in the dark overnight, and subjected to particle bombardment using a Biolistic PDS 1000/He System (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer’s instructions. The meristems were then allowed to germinate and were grown for 3–7 days before transfer to a selection medium containing 70 mg/L kanamycin. The explants were subcultured every 2 weeks and kanamycin concentrations gradually elevated to 140 mg/L; all controls died at 100 mg/L. The surviving plantlets were grafted to CCRI 12 plants and the regenerated, primary, transformed plants designated as T 0 plants; seeds from self-fertilization of T 0 plants were used to raise T 1 and subsequent progeny.

Analysis of transgenic plants by PCR and Southern blotting

T 0 plant leaves were treated with 1,500 mg/L kanamycin and 18 of 31 plants appeared positive for transformation. The presence of Bn-csRRM2 in these plants was determined by PCR analysis carried out using a PCR Screening Kit (TaKaRa Bio Co., Dalian, China), a forward primer (5′-AGTCGTGGATGCGGGTTTGTTA-3′), and a reverse primer (5′-GCAAGGCGATTAAGTTGGGTAA-3′). PCR conditions involved denaturing at 95°C for 5 min followed by 32 cycles of 40 s at 95°C for denaturation, 40 s at 58°C for annealing, 45 s at 72°C for elongation, and finally incubation at 72°C for 5 min. The expected PCR product size was about 210 bp.

The transgenic status of the selected plants was confirmed by Southern blot analyses showing positive reactions from the PCR. A DIG High Primer DNA Labeling and Detection Starter Kit I (Roche Applied Science, USA) was used for the labeling and hybridization process, following the standard protocol. Here, HindIII digested cotton genomic DNA (30 μg) and digoxigenin-labeled DNA Marker (Lambda DNA/EcoRI + HindIII Marker, Fermentas International, Inc., China) were electrophoresed in 1% agarose gel and transferred onto a Hybond-N+ nylon membrane (Amersham Biosciences, Buckinghamshire, UK) according to the standard protocol; the probes for Southern hybridization were fragments of the Bn-csRRM2 coding sequences.

Determination of fiber growth parameters

Mature bolls of each plant were collected and counted and, for weight determination, the lint and seeds from each plant’s bolls were separated and weighed. Raw cotton parameters, such as fiber length, strength, uniformity, and micronaire (cotton fineness) value, were assessed using a High Volume Instrument HFT 9000 (Premier, India) according to the USDA mode (Ibrahim 2010; Wang et al. 2010).

Microarray

Transgenic and wild-type cotton plants were grown under the same conditions and the leaves of day 25 and 45 plants harvested for microarray analysis. RNA samples were isolated from three replicates using Trizol (Invitrogen, USA) as described by the manufacturer. Microarray analyses were carried out using an Agilent Cotton Gene Expression Microarray (G2519F-022523, Agilent Technologies, USA), scanned on an Agilent Technologies Scanner (G2505C), and data collected using Agilent Feature Extraction software (version 10.7.1.1). Comparisons were made between transgenic samples and their corresponding wild-type samples and all microarray data were deposited in a public database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE26445). Genes with two-fold differences in expression and supported by a probability of ≤0.05 were considered significant. Data was analyzed using GeneSpring GX Software (Agilent Technologies) and Gene Ontology Enrichment Analysis Software Toolkit (http://omicslab.genetics.ac.cn/GOEAST/php/agilent.php).

Results

Expression of Bn-csRRM2 in cotton

To investigate the potential effect of Bn-csRRM2 in cotton, pBin438-Bn-csRRM2 was introduced into the shoot-tip meristem tissues of the elite cotton variety CCRI 12 by particle bombardment and, based on kanamycin resistance and PCR analysis for the presence of Bn-csRRM2, 12 independently transformed plantlets were obtained. Four of these plants (L008–L011) were randomly selected for further study and Southern blot hybridization indicated that L008–L011 had different Bn-csRRM2 copy numbers and insertion sites, confirming them as arising from independent transformation events (Fig. 1). Seeds of L008–L011 (T 0) were collected separately for further experiments.

Fig. 1
figure 1

Southern blot hybridization of Bn-csRRM2 in cotton. Binary vector pBin438-Bn-csRRM2 was introduced into cotton by particle bombardment; 12 independently transformed plantlets were obtained by kanamycin-resistant screening and PCR analysis for Bn-csRRM2 presence; four plants (L008–L011) were randomly selected for Southern blot hybridization (fragment of Bn-csRRM2 probe used to hybridize genomic DNA); the result indicated that L008–L011 had different Bn-csRRM2 copy numbers and insertion sites, confirming them to be from independent transformation events

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Expression of Bn-csRRM2 increased plant size

The T 1 plants of L008–L011 displayed visible increases in plant size (L008, Fig. 2e). Statistical analysis of the T 2 and T 3 generations of these four transgenic lines showed that kanamycin-resistant and Bn-csRRM2 PCR-positive plants (109.8 ± 6.8 cm, n = 14) were significantly larger than control CCRI 12 plants (82.2 ± 5.5 cm, n = 5, P < 0.01), whereas the Bn-csRRM2 PCR-negative plants without kanamycin-resistance (83.4 ± 7.2 cm, n = 8) were similar to control CCRI 12 (P = 0.75, Online Resource 2). These data indicated that enlarged plant size indeed resulted from Bn-csRRM2 ectopic expression.

Fig. 2
figure 2

Bn-csRRM2 affected sizes of plants and multiple cotton organs. Controls on left or top of each panel; a flower, 1 day post-anthesis (DPA), bar = 1 cm; b ovary, 1 DPA, bar = 5 mm; c boll, bar = 1 cm; d cystigenic valve, bar = 5 mm; e whole plant, full-bloom stage, bar = 20 cm; f sepal, 1 DPA, bar = 2 cm; g cotyledon, fully expanded, bar = 1 cm; h leaf, 1st leaf pair photographed when 2nd leaf pair emerged, bar = 5 cm

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Bn-csRRM2 induced increased organ size

Expression of Bn-csRRM2 in cotton did not alter the general organ morphology, but led to significant enlargement of multiple organs, including flowers, ovaries, cystigenic valves, bolls, sepals, cotyledons, leaves, stems (Fig. 2), and seeds. Cotton seed enlargement resulted in significant increases in average seed weight, ranging 6–24% greater than controls (Fig. 3a). Increases in boll size were associated with significant increases in lint weight/boll, ranging 9–40% greater than controls (Fig. 3b). At the same time, the bolls/plant also showed a marked increase, ranging 12–34% greater than controls (Fig. 3c). Consequently, the increase in lint yield/plant was significant, ranging 35–66% greater than controls (Fig. 3d).

Fig. 3
figure 3

Bn-csRRM2 increased lint yield and seed size. a Average weight of 100 randomly selected seeds: L008, 117.0 mg; L009, 111.1 mg; L010, 100.0 mg; L011, 112.4 mg; and control CCRI 12, 94.3 mg. b For each line, five plants for lint weight/boll; means ± SD: L008, 2.35 ± 0.14 g; L009, 3.00 ± 0.08 g; L010, 2.63 ± 0.37 g; L011, 2.52 ± 0.14 g; and control, 2.15 ± 0.08 g. c For each line, 10 plants for bolls/plant; means ± SD: L008, 18.60 ± 2.07; L009, 17.80 ± 1.32; L010, 16.80 ± 2.04; L011, 20.10 ± 2.81; and control, 15.00 ± 1.56. d Lint weight/plant calculated by lint weight/boll times bolls/plant: L008, 43.71 g; L009, 53.40 g; L010, 44.18 g; L011, 50.65 g; and control, 32.25 g

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Bn-csRRM2 induced increased cell size

A change in organ size can reflect an alteration in the size or number of cells, or both. In transgenic rice which constitutively overexpressed Os-csRRM2, histological analysis showed that the increase in organ size resulted from the increase in cell size (Hong et al. 2007). In our study, expression of Bn-csRRM2 also led to changes at the cellular level. The cotyledon petiole cells of the transgenic plants were noticeably larger than the same cell type in control plants (Fig. 4d, e). Similarly, cells of other organs were clearly larger than controls (data not shown); the most obvious was increased cotton fiber cell size. Photoelectric measurement showed that the cotton fiber length increased considerably (Table 1; Fig. 4a), and was associated with increased fiber strength (Table 1). Longer fiber length and higher strength are both highly desirable attributes.

Fig. 4
figure 4

Bn-csRRM2 affected cotton cell size. a Fiber length of transgenic (bottom) was significantly longer than control CCRI 12 (top), bar = 8 mm; transgenic pollen grain (c) was bigger than control (b), bars = 100 μm; cross-sections of cotyledon petiole (cut from basal part of fully expanded cotyledons) showed transgenic cells (e) larger than control cells (d), bars = 320 μm

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Table 1 Bn-csRRM2 improved fiber quality
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Pollen is the flower structure which transports the male gamete to the ovule and within the exine of a binucleate pollen grain are a generative nucleus and a large vegetative cell. The latter accumulates abundant stored metabolites required for rapid pollen tube extension, while the diminutive generative cell is enclosed by vegetative cell cytoplasm and contains relatively few organelles and stored metabolites (Bedinger 1992). Thus, pollen size fairly reflects cell size. To investigate whether Bn-csRRM2 increased pollen size, pollen cross-sectional areas were determined by the Scanning Image Pixels Method (Ohto et al. 2005). Three each of transgenic cotton and control CCRI 12 plants were analyzed, with pollen grains from anthers deposited onto a glass slide and observed immediately by light microscopy. The cross-sectional areas of at least 30 pollen grains were measured and averaged for each plant. Statistical analysis showed that the transgenic pollen was significantly larger than control pollen (P < 0.01, Online Resource 3, Figs. 4b, c).

Taken together, these results demonstrated that the increase in transgenic organ size was due in part to increased cell size. In most crop production, high yield is the most important goal and sources of yield include cell numbers, size, and mass (Frary et al. 2000). The present observations that constitutive expression of csRRM2 enhanced the sizes of multiple cell types could have high practical value.

Discussion

Many reports on FCA have related to its roles in floral development (Macknight et al. 1997, 2002). It negatively regulates its own expression by promoting cleavage and polyadenylation of its pre-mRNA, which causes the production of a truncated, inactive transcript at the expense of the full-length FCA mRNA, thus limiting the expression of active FCA protein (Quesada et al. 2003). The overexpression of csRRM2 may affect FCA autoregulation. As the manipulation of FCA did not result in similar phenotypes, cell-size regulation should be a new function of csRRM2 (Macknight et al. 1997; Furner et al. 1996).

RRM domains are abundant in all life kingdoms (Lorkovic and Barta 2002) and are known to recognize and bind sequence-specific RNA or DNA (Burd and Dreyfuss 1994; Maris et al. 2005). By associating with different protein domain types, RRM domains can modulate their RNA-binding affinity and specificity, thus diversifying their biological function (Maris et al. 2005; Eulalio et al. 2009). Moreover, besides nucleic acid binding, they participate in diverse protein–protein interactions (Trzcińska-Daneluti et al. 2007). FCA possesses two RRM domains, which have been reported to be involved in polyadenylation site selection (Simpson et al. 2003), chromatin silencing of single and low-copy genes, and interaction with the canonical small interfering RNA-directed DNA methylation pathway for regulating common targets (Baurle et al. 2007; Baurle and Dean 2008). In the present study, Bn-csRRM2 was observed to be involved in cell-size regulation. For multicellular plants, average cell size not only varies with genotype but also varies between different tissues within the same genotype, which appears to suggest that there is specificity in cell-size regulation for individual cell types (Yi et al. 2010). Thus, the finding here that the sizes of several cell types were similarly increased by ectopic Bn-csRRM2 expression in G. hirsutum was unexpected and suggested that csRRM2 may be a conserved and ancient cell-size regulator and that this regulation may exist at two levels in higher plants, one at the individual cell type level and another at a global level, affecting most cell types.

Eukaryotic RRM proteins are known to modulate gene expression by participating in different post-transcriptional events (Maris et al. 2005). Agilent Cotton Gene Expression Microarray was used to identify the genes differentially expressed in these Bn-csRRM2 cotton plants. Comparisons were made between transgenic cotton and control CCRI 12 at day 25 and 45. The results showed that 67 and 277 genes, respectively, were significantly regulated (Online Resource 4 and 5). Gene ontology (GO) classification revealed that cytosolic large ribosomal subunit (GO: 0022625) was the top-represented GO category (P = 0.02) in the 45-day group. Furthermore, 16 genes showed regulation in both developmental stages and were considered to be closely related to the Bn-csRRM2-induced phenotypes (Online Resource 6), but the involvement of these in common genes in cell-size regulation await future experimentation. Among these genes, two 60S ribosomal protein genes (ES800645 and ES843981), whose amino acid sequences are strongly similar (94.6 and 94.2% identity, respectively) to Arabidopsis thaliana 60S ribosomal protein L35a-3 (TAIR: AT1G74270) and L23a-1 (TAIR: AT2G39460), were both downregulated in transgenic cotton. It has been reported that deletion of genes encoding 60S subunit proteins or processing factors or treatment with a small molecule, which all inhibit 60S subunit biogenesis, are each sufficient to significantly increase replicative life span in yeast (Steffen et al. 2008). The downregulation of the two 60S ribosomal protein genes might also extend the cell cycle. At the same time, ES816367, whose amino acid sequence is moderately similar (79.8% identity) to Arabidopsis thaliana DEHYDROASCORBATE REDUCTASE 2 (DHAR2; TAIR: AT1G75270), was also downregulated. It has been reported that ascorbate stimulates cell cycle activity in competent cells, while the oxidised form, dehydroascorbate, blocks normal cell cycle progression (Potters et al. 2002), indicating that the redox state of the ascorbate–dehydroascorbate pair is a specific regulator of cell division (De Pinto et al. 1999). Downregulation of DHAR2 might increase the level of dehydroascorbate, which, in turn, blocks cell cycle progression. These lines of evidence suggested that Bn-csRRM2 overexpression might inhibit cell cycle progression and, consequently, result in the increase in cell size.