Enhanced Expression of Plasma Membrane Intrinsic Protein 2 Improves Cotton Fiber Length and Potential Economic Viability of Gossypium Arboreum

Background: Gossypium arboreum is a cotton crop native to tropical and subtropical regions that are naturally resistant to Cotton Leaf Curl Virus (CLCuV). However, its cultivation is unfavorable due to the lower quality and shorter ber length of cotton when compared to the market leading Gossypium hirsutum. Plasma membrane intrinsic protein 2 (PIP2) is an aquaporin responsible for the transport of water and small molecules across cellular membranes. This uid transport inuences cell elongation and cotton bre development. Hence, increased PIP2 expression may yield plants with enhanced ber qualities including length. Methods and Results: To test this hypothesis, G. arboretum was transformed with a PIP2 gene construct (35SCpPIP2)using the Agrobacterium-mediated shoot apex cutting method. Relative expression of the CpPIP2 gene in transgenic plants increased up to 35-fold when compared with non-transgenic controls. Transgenic plants displayed a corresponding increase of staple length (up to 150%) when compared with non-transgenic controls. Transgene integration was examined using FISH and karyotyping and revealed the presence of a single transgene located on chromosome 6. Conclusion: Since G. arboreum is naturally whitey and CLCuV resistant, this improvement of ber length evidenced for CpPIP2 transgenic plants renders their crop production more economically viable. Primer The gene-specic primer 5’-CCACCCCTACTCCAAAAATG-3’ reverse primer an initial denaturation at 95 (cid:0) C for 5 min, and then performed at 95 (cid:0) C for 30s, 51 (cid:0) C for 45s,and 72 (cid:0) C for 1min, for a total of 35 cycles. The forward primer 5’-AGGAATTGCTTGGGCTTTCG-3’ and reverse primer 5’-TGGAATGCCTTCACGAATCC-3’ were used for gene amplication. The qPCR analysis was run on PikoReal real-time PCR system (Thermo Fisher Scientic). CLCuV,cotton leaf curl virus; dNTPs, deoxynucleotide triphosphates; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GOT%, ginning out turn percentage; PCR, polymerase chain reaction; PIPs, plasma membrane intrinsic proteins; TP, transgenic plant.


Introduction
Cotton is the primary source of ber for garment production within the textile industry. Cotton crops (also known as white gold) are cultivated globally but especially in the tropics and subtropics including Pakistan; the fourth largest cotton producer worldwide [1].Gossypium arboreum(desi cotton) (Family Malvaceae)is native to India and Pakistan and is adiploid (2n) cotton species [2]. Gossypium hirsutum is a tetraploid product of hybridization between the diploids G. arboreum and G. raimondii. G. hirsutum is the most widely cultivated cotton species and is utilized for ≈ 90% of the world's cotton production. However, G. hirsutum is vulnerable to infection by cotton leaf curl virus (CLCuV). By comparison, G. arboretum carries genes that confer resistance to CLCuV infection [2,3],yet it is only responsible for ≈ 2% of total world cotton production. This difference in the cultivation trends between these two cotton plants in part re ects the bene ts of longer ber length: upto 30 mm for G. hirsutum compared to ≈ 15 mm for G. arboreum [4,5]. Fiber length in uences the strength, evenness, and the ability to spin bers to produce the nal yarn. Longer bers can be more e ciently spun into yarn, hence there is a bene t to cotton breeding that increases ber length, if achieved without a compromise in ber quality.
Calotropis procera (Milkweed)(Family Asclepiadaceae)is a wild perennial shrub, native to parts of Asia and Africa. C. procera is cultivated in South America and the Caribbean Islands for ber production and produces cotton of relatively high tensile strength [6][7][8].C. procera produces seed bers that are silky white and relatively long (≈ 30-45 mm length) and utilized for pillow stu ng, weaving of cloth, and surgical cotton wool [8,9]. The relatively high C.procera ber length in part relates to the expression of aquaporin proteins that function in cell expansion [3,[10][11][12][13].
Aquaporins are small integral plasma membrane proteins responsible for the transport of water and small molecules across the cell membrane. This uid transport in uences cell elongation and bre development [3]. Plasma membrane intrinsic proteins (PIPs), a subclass of aquaporins, play a role in ber cell elongation by maintaining turgor pressure. PIPs constitute the largest group of aquaporins and are further subdivided into PIP1 and PIP2 proteins.PIP2 proteins display higher water transport activity than PIP1 and improve a plant's tolerance against drought by maintaining osmotic balance, water use e ciency, and water retention [14][15][16][17].
Therefore, the potential exists for exploitation of aquaporins such as PIP2,through their incorporation into other cotton plant species to potentially improve ber length and ber quality [18].The present study details a biotechnological approach to attempt to improve cotton berporpertiesofG.arboreumFBD-1 (Four Brothers Desi-1), a local Pakistani G. arboreum cotton variety. G. arboreum FBD-1 was transformed with a PIP2gene construct isolated from C. procera. Cotton ber properties including ber length were then compared with non-transgenic control plants.

Source of plant material
The G.arboreumFBD-1variety of cotton was selected basis on its high germination percentage and yield.
Seeds of FBD1 were obtained from the Four Brothers Group of Pakistan on a research collaboration basis. Recombinant plasmid pGA482 containing the 35SCpPIP2 gene construct was provided by Forman Christian College, Lahore, Pakistan.

Computational analysis of the PIP2 construct
The nucleotide sequence of theCpPIP2 gene construct (that was used to transform G.arboreum) was translated into a protein sequence using the online tool EMBOSS-Transeq (https://www.ebi.ac.uk/Tools/st/emboss_transeq/). The molecular structure of PIP2 protein was then modelled from the amino acid sequence using the SWISS-MODEL Server (https://swissmodel.expasy.org/). Since there is not a 3D structure available for C. proceraPIP2 protein in the Protein Data Bank (PDB),a 3D structure of spinach aquaporin SoPIP2 (PDB ID: 4IA4) was used as a template for protein modelling. Structures of SoPIP2 and CpPIP2 were compared using the FATCAT server (http://fatcat.sanfordburnham.org/fatcat-cgi/cgi/fatcat.pl?-func=pairwise) to determine the con dence of the newly built protein structure.
Ampli cation of the PIP2 construct in E. coli and transformation of Agrobacterium tumefaciens The pre-cloned35SCpPIP2construct [11] was ampli ed in E. coli strain TOP10. The puri ed recombinant plasmid pGA482 containing the CpPIP2 gene was con rmed by polymerase chain reaction (PCR) ampli cation with the gene-speci c primers (forward:5'-CCACCCCTACTCCAAAAATG-3'; reverse: 5'-AATCCCACACCGCAGATAG-3'). The PCR mix was comprised of 1µL of plasmid, 2µL of 10x PCR buffer (Fermentas cat# B34), 2µL of 2mM dNTPs, 1µL of 25mMMgCl 2 , 2µL of each of forward and reverse primers (10pM) and 0.5 µL of Taq Polymerase enzyme 5U (Fermentas cat# EP0071) and deionized water to a 20µL nal reaction volume. PCR was performed at 95 Cfor 5min for 1cycle, and then 35 cycles of 95 Cfor 45s, 63 Cfor 45 s and 72 C for 1min, and then a nal ampli cation at 72 C for 10 min. The recombinant plasmid was then transformed into Agrobacterium tumefaciens strain LBA4404 by electroporation using an electroporator (Bio-Rad, California, USA). The transformed A. tumefaciens cells were then incubated on ice for 2 minutes and diluted with 1 mLYeast extract peptone (YEP) broth and incubated at 30 C for 3 hours at 200 rpm. Then, 100µLof cultured cells were plated on YEP medium supplemented with 50µg/mL kanamycin and grown at 30 C.Transformed A. tumefaciens colonies were isolated and screened again to con rm the gene insert by PCR, using the gene-speci c primers.
Transformation of the CpPIP2 gene into G. arboretum Cotton seeds were de-linted with concentrated sulphuric acid, and then surface sterilized with0.1% HgCl 2 and 0.1% sodium dodecyl sulphate and rinsed thoroughly with sterilized distilled water. The sterilized cotton seeds were then germinated in the dark at 30 C. The germinated embryos of 30-36 hr were used for Agrobacterium-mediated transformation as per the method of Rao et al.(2011) [19].The explants were incubated with Agrobacterium (overnight grown and suspended in Murashige and Skoog(MS) broth) for 30-60 min at 28 C in the dark. The shoot apex explants were then blot-dried on sterile lter paper and placed into semi-solid MS (minimal growth medium) plates supplemented with cefotaxime (100µg/mL). Several untreated shoot apexes injured similarly were Agrobacterium-treated and plated as controls. The healthy plantlets were then transferred to test tubes containing MS and supplemented with 50µg/mL kanamycin and 100µg/mL cefotaxime and allowed to grow under 60μE m −2 s −1 light for 16/8hrlight/dark cycle, for in vitro growth at 28 C+ 2 C for 2-3months.

Acclimatization of tissue-cultured cotton plants
After attaining a height of approximately 6inches,G. arboreum seedlings in glass tubes were transferred to sterilized soil pots of 6-inch diameter. They were then covered with polythene bags for gradual acclimatization to a culture room temperature environment, starting from 15 min and gradually increasing to a full day in a culture room. The plants were further acclimatized to sunlight for hardening in a similar fashion as above and then shifted to a eld tunnel.

Fluorescent in situ Hybridization (FISH)
FISH was employed to determine the chromosomal location of the CpPIP2 gene in transgenic plants as described by Gul et al. (2020) [20].

Statistical analysis
Statistical comparisons between plants were performed by one-way analysis of variance (ANOVA), with Dunnett's multiple comparison test, using GraphPad Prism (version 7)(GraphPad Software Inc., San Diego, California, USA).Signi cant differences were set at a p-value of < 0.05. Data presented as histograms represent experimental means ± SD.

Computational Analysis of PIP2
To examine the potential bene t of incorporation of the aquaporin PIP2 into G. arboretum, a molecular modelling approach was rst undertaken. A 3D protein model of the transformed CpPIP2 nucleotide sequence was built via the SWISS-MODEL server, using spinach (Spinacia oleracea) aquaporin SoPIP2 (PDB ID: 4IA4) as a template. The resulting 3D model showed that this protein is tunnel-shaped with six transmembrane domains (Figures 1A & 1B).A FATCAT server calculation showed that there was an ≈86% similarity of CpPIP2with the aquaporin SoPIP2 (PDB ID: 4IA4). Indeed, a total of 241 amino acid residues were perfectly aligned between the two proteins and the root mean square distance of the aligned regions was 0.37 [21]. The superimposition of the two structures ( Figure 1C) con rms the pore-forming shape of the CpPIP2 gene product and suggests that after incorporation into a recipient plant (G. arboretum), the protein may be useful for improved water and ion ux; traits conducive to improved ber elongation.

Transformation of the CpPIP2construct
Transformation of G. arboreum with the CpPIP2 gene was carried out using an A. tumefaciens strain, LBA4404. A total of 4055 shoot apices of the germinated embryos were injured and co-cultivated with A. tumefaciens containing the CpPIP2 gene. Only 231 plantlets survived in MS-plates during the cocultivation stage, as the majority of plantlets died from either a fungal infection or an inability to develop roots or shoots. The healthy plantlets that survived the kanamycin selection for approximately two months were regarded as putatively transformed plants and were shifted to soil pots. The plants in soil pots (54) were then transferred to eld tunnels after acclimatization. The transformation e ciency for G. arboreum was calculated as 1.3% (Table 1) according to the formula: To verify the transformation and production of transgenic cotton plants, ampli cation of the 844bp PCR insert corresponding to the CpPIP2 gene was undertaken, as shown in Figure 2.  TP1, TP2, TP3, TP4, TP5, TP6, TP7 and TP8,respectively ( Figure 3).

Morphological assessment of cotton bers
An assessment of ber quality was undertaken to consider the effects of the CpPIP2 gene transgene on cotton properties. PIP proteins are involved in trichome elongation and stress tolerance, hence ber length, strength, micronaire value, uniformity index, and GOT% were analyzed. Expression of the CpPIP2 gene in transgenic cotton plants produced a signi cant increase of ber length but was without effect on the other ber characteristics when compared with non-transgenic control plants (Figures 4 and  5A).Notably, an increase in ber length was observed for all transgenic lines compared to non-transgenic controls ( Figure 5A). The transgenic plant, TP1, produced the greatest increase in ber length compared to the non-transgenic controls, followed by TP5 and TP6 (Figure 5 A, B). These results are consistent with the mRNA expression data for these transgenic plants (Figure 3), indicative of a direct correlation between gene and protein expression.

Localization of CpPIP2chromosomeal integration
Fluorescent insitu hybridization (FISH) was undertaken for the TP1 transgenic plant. The uorescent signal was localized to chromosome 6 in the hemizygous form, con rming that theCpPIP2 gene had been successfully integrated into the cotton genome ( Figure 6).

Discussion
Rising global demand for cotton has highlighted the need to improve both cotton ber quality and length.
This can be achieved through the biotechnological manipulation of cotton plants. Although G. hirsutum has the market share of cotton production, it is vulnerable to white y and CLCuV infestation, leading to crop losses [22,23].To exploit the inherent resistant nature of G. arboreum against both white y and CLCuV but with potentially improved cotton ber traits, the CpPIP2 gene encoding a PIP2 aquaporin was introduced into G. arboreum.
Aquaporins comprise a diverse protein family responsible for the transport of water and solutes in plants and animals [14,24].This functionality is re ected by the formation of barrel-like membrane pores, such as that for PIP2 (Fig. 1). PIP2 proteins recruit PIP1 proteins from the endoplasmic reticulum to the plasma membrane to increase water permeability [14]. In this study, G. arboreum was successfully transformed using an Agrobacterium-mediated shoot apex cut method [19] to produce transgenic plants with elevated PIP2 expression. Although transformation e ciency was relatively low (Table 1), it was still in keeping with other transgenic plant studies [25,26]. Moreover, relative CpPIP2gene expression of transgenic plants was high (Fig. 3).
The incorporated transgene,CpPIP2,is controlled via aconstitutively active35S promoter. Hence, all successfully transformed plants displayed increased PIP2 expression. From the analysis of 8 separate plants (TP1-TP8), identi cation of the highest expressing transgenic plants (TP1, TP5 and TP6) was accomplished (Fig. 3).The utilization of the 35S promoter to driveCpPIP2expression,(by comparison to thetrichome-speci c promoter (GhLTP3),has previously been ulitized for gene expression studies in transgenic tobacco plants [11].Relative PIP2expression for transgenic plants was 15-38-fold higher than non-transgenic controls. For the transgenic plant, TP1, that displayed thehighest expressional increase of PIP2, an increase o ber length of 150% (15mm to 22mm) was observed.This bene cial increase ofcotton ber length for transgenic plants arose without change to other integral ber properties. Indeed, there were no signi cant differences between control or transgenic plants for ber strength, micronair value ( ber neness and maturity), uniformity index, or GOT% (Fig. 4).
In summary, G. arboreum is not a preferred cotton cultivar due to its shorter ber length. However, it has a natural resistance to several chewing and sucking insect infestations including the notorious white y, the biological vector responsible for CLCuV transmission [22,27]. Hence, this genetically modi ed G. arboretum, with an improved ber length without loss of ber quality, could potentially improve the economic viability of G. arboreum via increased cotton yield. data analysis, to produce the nalized manuscript. AAS reviewed and approved the article for submission. All authors read and approved the nal manuscript.       FISH and karyotyping of CpPIP2 transgenic plants. The CpPIP2transgene was localized to chromosome number 6 in the diploid cotton genome as con rmed by uorescence (red arrow).