Dear Editor,

Proper development of plant roots is critical for primary physiological functions, including water and nutrient absorption and uptake, physical support, and carbohydrate storage (Zhang et al., 2010). Crop root systems act as the key organ for sensing and response to abiotic and biotic stresses. Previous studies of crop root system development suggest that increased lateral root formation (LRF) could stimulate whole plant development, and ultimately increase crop yield. Cotton (Gossypium spp.) is the largest natural fiber crop, and its fiber is used as an important material around the world. Cotton has a typical taproot system which includes main and lateral roots; function of the root system is strongly affected by lateral root development. Improvement of lateral root formation could increase total root surface areas, potentially promoting growth of the whole cotton plant, and ultimately increasing fiber yields, especially under adverse conditions of drought and low soil fertility. Breeding new cotton lines with increased LRF traits, or larger root surface area, will not only expand the arable cultivation range (e.g., arid and low fertility soil could be planted), but also will increase fiber yield, resulting in eventual applications that promote development of the entire cotton industry production chain.

Recent studies demonstrated that the enzymatic biosynthesis pathway for nitric oxide synthase (NOS), which catalyzes the synthesis of nitric oxide (NO) from arginine (ARG), is the primary source for root NO, a regulator of plant root development (Correa et al., 2004). Interestingly, NOS enzyme activity could be inhibited by an increase in the activity of arginase, which competes for ARG substrate. In Arabidopsis, reduced arginase activity in argah1-1 and argah2-1 mutant lines increased NO accumulation, and resulted in formation of lateral and adventitious roots. Specifically, the total lateral roots per primary root in the mutants were twice the number observed in the wild type. Our previous studies showed that overexpression of the rice arginase gene (OsARG) in upland cotton (Gossypium hirsutum) significantly decreased root NO accumulation, and inhibited lateral root development (Meng et al., 2015). Plant arginase thus plays an important role in regulation of lateral root formation, and artificial manipulation of its expression may promote an ideal LRF in cotton.

The clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) system is a high efficiency gene editing technique which has been widely used to create transgenic mutant plants (Chen and Gao, 2014). The system consists of a Cas9 endonuclease and a guide RNA which contains a 20 nt specific sequence that is complementary to target sites in the genome (Zhang et al., 2016). Several successful studies using the CRISPR/Cas9 system have been reported in wheat, tobacco, rice and many other crops. However, to our knowledge no successful application has yet been reported in upland cotton. Here, we use CRISPR/Cas9 genome editing technology to knock out the cotton arginase gene (GhARG) to improve LRF. The goal is to create new, high-yield, transgenic cotton lines for germ line breeding stock that carry valuable traits, such has high adaptability to nutrient- and water-limited, high saline soils.

Upland Cotton is an allotetraploid plant with two chromosome types (A- and D-genome), which include several, chromosomally integrated, multi-copy genes. The cotton protoplasts are hard to harvest because of the high polyphenols and pigments content. Low transformation efficiency of cotton protoplasts limits our ability to validate the editing efficiency of CRISPR/Cas9 for target sites in cotton. To mitigate this limitation, we designed a CRISPR/Cas9 construct for which agrobacterium-mediated transformation could be used to directly transform cotton tissue. To efficiently and accurately screen for transgenic cells and calluses during the plant tissue culture stage of the transformation process, we utilized green fluorescent protein (GFP) and neomycin phosphotransferase gene (NPTII) marker genes. Marker-selected calluses with the highest edit efficiency were confirmed by PCR and used for seedling regeneration. The primers used for vector construction are listed in Table S1 in Supporting Information.

Given that there are two, highly similar, orthologous, cotton arginase genes (GhARG), Gh_A05G2143 andGh_D05G2397, in the A- and D-chromosomes, respectively, we designed two, independent sgRNAs (driven by the NtU6 promoter) with 20 nt overlapping a homologous coding region in the first exon of each gene (Figure 1A). We then transformed these constructs into upland cotton R18, a transgenic acceptor variety bred from the Coker 312 cotton, which is, globally, a main transgenic acceptor germ line.

Sequencing and PCR screens of genomic DNA showed that most calluses were successfully edited at the target site with non-homologous recombination repair at the targeted site. Gel electrophoresis further showed that the edit frequency of sgRNA1 and sgRNA2 ranged from 10%–98% and 10%–75%, respectively (Figure 1C, Figures S1 and S2 in Supporting Information). Comparison of the edit efficiency between the two transgenic calluses showed that the efficiency for sgRNA1 calluses was higher than for those of sgRNA2. Five out of fifteen sgRNA1 transgenic calluses had an edit efficiency from 80%–98% (Figure 1C, sgRNA1 lane 5, 6, 7, 9 and 14), while only three out of fifteen sgRNA2 calluses had an edit efficiency higher than 70 percent (with a high of 75%) (Figure 1C, sgRNA2 lane 1, 7 and 13). Calluses produced using sgRNA1 were used to regenerate and screen mutated plants, because of the high edit efficiency for this construct.

After T0 plantlets were regenerated from sgRNA1 callus, genomic DNA was used to identify the edit type, or target site indels, of each mutant. Alignment results indicated that both Gh_A05G2143 and Gh_D05G2397 (Figure 1D) had several target site edit variants (Figure S3 in Supporting Information). As shown in Figure 1D, the edit type of mutant Lines 6 and 9 in the T0 generation of sgRNA1 plants could be divided into 7 and 3 types respectively. Edits in these lines ranged from 1 bp insertions to 1 to 44 bp deletions. Sequence analysis of T0 offspring plants (Figure S4 in Supporting Information) also demonstrated that both Gh_A05G2143 and Gh_D05G2397 were knocked out, and that these genotypes and traits were passed on to offspring.

To investigate the effects of these edits on lateral root formation, we planted T1 transgenic seedlings from Line24 (offspring of Line6) and Line28 (offspring of Line9) on low nitric medium, 1/2 MS medium and 1/2 MS medium with 4-fold nitric medium (high nitrogen content). Under normal growth conditions, both Line24 and Line 28 showed clear promotion of LRF in high nitric medium. Examination of lateral root traits in two GhARG lines, Line 24 and Line 28, showed a 25% and 46% increase in the number of laterals roots, and 52% and 74% in total root surface area, respectively, compared with our wild-type controls. Similar phenotypes were observed following growth on low nitric medium (Figure 1E and F). Further analysis of enzyme activity (arginase and NOS) and NO content (determined by nitrate/nitrite (NOx) content revealed that arginase activity was sharply decreased in the transgenic seedlings (Figure S5 in Supporting Information), followed by significant elevation of NOS activity (Figure S6 in Supporting Information) and NOx content (Figure S7 in Supporting Information). These data suggest that edits to the first exon of GhARG caused coding sequence mutations that inactivated the arginase enzyme, resulting in partial or complete loss of arginase function in transgenic seedlings. Decreased arginase activity in cotton seedlings stimulates NOS activity, generating a feedback loop that induces ARG to synthesize more NO, ultimately promoting lateral root development.

In conclusion, we successfully knocked out GhARG genes on both the A- and D-chromosomes of allotetraploid upland cotton using the CRISPR/Cas9 system. The CRISPR/Cas9-mediated mutations significantly improved lateral root system development under both high- and low-nitric conditions. Due to its strong, lateral root system, we propose that genetically modified cotton can be adopted for cultivation in a wide range of soil types, while yielding more commercially useful fiber. Given that most crops are polyploid, the application of CRISPR/Cas9 for editing polyploid plant genomes becomes increasingly attractive for molecular plant breeders. Our study is particularly valuable in light of the paucity of successful studies on the creation of genome-edited cotton to improve agronomic traits. Our research demonstrates that the CRISPR/Cas9 system can be utilized with high efficiency in polyploid crops to edit multi-copy genes.