The vast majority of the world's cotton varieties produce white fibre, which is then
coloured with synthetic dyes during textile processing to fulfil the diverse needs
of consumers. This results in potential environmental pollution and is detrimental
to human and animal health. Although there exists naturally coloured cottons, mostly
in brown and green, their poor agronomic performance and suboptimal fibre quality
have severely hampered their widespread applications in cotton breeding program. Therefore,
there is an impetus to create multicoloured fibre through synthetic biology biotechnology
by leveraging the biochemical processes of colour formation and associated regulatory
genes in cotton.
Pigment accumulation typically results in colourful phenotypes in various plant tissues.
Proanthocyanidins or lignin are the pigments responsible for the brown and green cotton
fibre. Betalain and anthocyanin are two primary groups of red pigments that are found
in the vacuoles of plant cells. The former, present in dragon fruit (Hylocereus undulatus
Britt) and sugar beet (Beta vulgaris), is derived from the tyrosine synthesis pathway
through three sequential catalytic steps (Polturak and Aharoni, 2018), whereas the
latter requires the engineering of additional 13 or 14 genes (Zhu et al., 2017). In
Arabidopsis, rice, and tobacco, co‐expression of a P450 oxygenase (CYP76AD1), L‐DOPA
4,5‐dioxygenase (DODA), and Glucosyl Transferase (GT) resulted in considerable betalain
accumulation, which results in vibrant red colour in various tissues of transgenic
plants, respectively (He et al., 2020). However, it remains unknown if the betalain
pathway can be engineered into cotton plants to alter the colour of cotton fibre without
compromising fibre quality.
To generate cotton plants with colourful fibre, three genes, including CYP76AD1, DODA,
and GT from B. vulgaris were cotton codon optimized. Three transgene expression cassettes
(Figure 1a) termed “Ruby” by He et al. (2020), each driven by either a 2x35S CaMv
constitutive promoter (hereafter referred to as 35 S‐RUBY) or a fibre‐specific E6
promoter (hereafter referred to as E6‐RUBY), are separated by two 2A self‐cleavage
peptide‐encoding genes (Sharma et al., 2012; Figure 1b). Multiple independent transgenic
upland cotton (Gossypium hirsutum L.) plants overexpressing Ruby were generated using
a high‐yielding and high‐fibre‐quality modern elite variety, Zhongmian49, as a donor
plant (Ge et al., 2022; Figure 1c). PCR analysis detected that gene expression level
varies among different T0 plants, and two 35 S‐RUBY lines and one E6‐RUBY line with
higher expression level of GT in immature fibres were selected to perform DNA blotting
analysis, showing the stable integration of transgene (Figures 1d,e). Segregation
analysis indicated that the transgenes could be inherited into the following generation
(Figure 1f). Single copy T2 homozygous transgenic plants with high gene expression
levels, together with wild‐type Zhongmian49 (WT), were grown under field conditions
in a confined environment to evaluate the agronomic performance of transgenic lines
(Figure 1g). All of the transgenic plants accumulated betalain, but the accumulation
patterns were different depending on whether the plants were co‐overexpressing 35 S
CaMv‐driven betalain genes or E6‐driven betalain genes. Whereas the WT control showed
a normal phenotype, the leaf, stem, boll, bract, flower, anther, and seed were all
purple or pink in the 35 S‐RUBY plants (Figure 1h). Notably, light pink colour was
discernible in immature fibres at 12, 27, and 42 days post‐anthesis (DPA), but it
faded away when fibre completely matured (Figures 1h,i). The colour phenotype of the
E6‐RUBY plants remained unchanged from that of the WT, apart from the immature fibres,
which showed pink in colour at 12, 27 and 42 DPA. (Figures 1g,h). Although the pink
colour did not persist in mature fibres in both E6‐RUBY and 35 S‐RUBY transgenic lines
due to the lower level of betalain in mature fibre compared to leaves and seeds (Figure 1j),
colour fibres were observed in near‐maturity transgenic cotton plants at 45 DPA after
freeze (−40 to −50 °C) or high temperature treatment (~ 40 °C), and the pink colour
was stably inherited from T0 to T2 generation (Figures 1k,l). As illustrated in Figures 1m,n,p,t,
the fibre length and strength of the transgenic fibres at 45 DPA and mature stage
were comparable to those of the WT fibre. Moreover, there was no difference in the
weight of cotton bolls between transgenic plants and control (Figure 1o). It is hence
apparent that there are no quality trade‐offs in the transgenic lines overexpressing
the betalain genes in either a constitutive or fibre‐specific manner. Considering
the superior fibre yield and quality of Zhongmian49, these transgenic plants with
an intrinsically vibrant pink colour maintained at near‐maturity may have the potential
to be used for commercial exploration.
Figure 1
Accumulation of heterologous betalains results in pink cotton fibre. (a) The introduced
betalain biosynthesis pathway. (b) Three key betalain genes, each driven by a 35 S
CaMv promoter or an E6 promoter, were co‐expressed in Gossypium hirsutum. (c) Phenotype
of the seedlings of WT and T0 transgenic lines. (d) Expression level of GT gene in
the immature fibres of T0 transgenic lines. (e) DNA blot analysis of T0 transgenic
lines. (f) Transgene inheritance to offspring. (g) Phenotypes of WT, 35 S‐RUBY or
E6‐RUBY transgenic plants. (h) Betalain accumulation in seed, bract, flower, and boll
in developing cotton plants. (i) Phenotype of mature fibre of WT and transgenic lines.
(j) Betalain content in the leave, seed, and mature fibre of 35‐RUBY plant. The colour,
fibre length and strength of freeze‐dried fibre at 45 DPA in WT, T0 and T2 transgenic
plants (k–n).Cotton boll weight (o), mature fibre length (p), uniformity index (q),
fibre strength (r), micronaire values (s) and elongation rate (t) were measured in
the transgenic lines and WT plants.
As betalains are most stable in the pH range of 4.0–6.0, they can be used in textile
processing without additional treatment to improve colour stability and uniformity.
Future research could be directed to elucidate the regulatory processes that underpin
the degradation of betalain in fully mature fibres and develop alternative strategies,
e.g., increasing the deposition of betalain in fibre secondary cell walls (Huang et al., 2021).
Moreover, the development of a robust synthetic biology biotechnology for cotton is
envisaged to expedite the generation of pure inbred lines with multiple desirable
features, bypassing the lengthy procedure of repeated crossing and backcrossing in
conventional breeding. Crossing HS2 with naturally coloured fibres led to enrichment
and diversification in fibre colours (Ke et al., 2022). It is plausible to assume
that more vibrant or versatile colours may be attainable by crossing E6‐RUBY with
the naturally coloured fibres. Overall, our findings demonstrate for the first time
that it is possible to produce natural pink colour fibre by engineering key genes
involved in betalain synthesis without compromising fibre yield and quality in cotton,
providing an environmentally and health‐friendly alternative to synthetic dye for
producing coloured cotton fibre.
Author contributions
F‐G. L., X‐Y. G designed the studies and wrote the manuscript. P.W., Y.W., Y‐L. Chen
and X.W performed the experiments.
Conflict of interest
The authors declare no conflict of interest.