A novel single nucleotide mutation of TFL1 alters the plant architecture of Gossypium arboreum through changing the pre-mRNA splicing

Abstract

A single nucleotide mutation from G to A at the 201st position changed the 5' splice site and deleted 31 amino acids in the first exon of GaTFL1. Growth habit is an important agronomic trait that plays a decisive role in the plant architecture and crop yield. Cotton (Gossypium) tends to indeterminate growth, which is unsuitable for the once-over mechanical harvest system. Here, we identified a determinate growth mutant (dt1) in Gossypium arboreum by EMS mutagenesis, in which the main axis was terminated with the shoot apical meristem (SAM) converted into flowers. The map-based cloning of the dt1 locus showed a single nucleotide mutation from G to A at the 201st positions in TERMINAL FLOWER 1 (GaTFL1), which changed the alternative RNA 5' splice site and resulted in 31 amino acids deletion and loss of function of GaTFL1. Comparative transcriptomic RNA-Seq analysis identified many transporters responsible for the phytohormones, auxin, sugar, and flavonoids, which may function downstream of GaTFL1 to involve the plant architecture regulation. These findings indicate a novel alternative splicing mechanism involved in the post-transcriptional modification and TFL1 may function upstream of the auxin and sugar pathways through mediating their transport to determine the SAM fate and coordinate the vegetative and reproductive development from the SAM of the plant, which provides clues for the TFL1 mechanism in plant development regulation and provide research strategies for plant architecture improvement.

Keywords: Determinate growth; Gossypium arboreum; Plant architecture; TFL1.

Fig. 1

The phenotype of determinate growth and indeterminate growth in dt1 and wild type Shixiya 1. a The plant architecture of the dt1 mutant and the wild type Shixiya 1 (Scale bar = 7 cm) at the flower bud stage. b–c The destiny of the shoot apical meristem (SAM) in the dt1 mutant and the wild type Shixiya 1. The white arrow indicates that the SAM became terminal flowers in the dt1 mutant (c) compared with the young leaf formed in the wild type Shixiya 1 (b) (Scale bar = 2 cm). d–e The fruit branch of the dt1 mutant and the wild type Shixiya 1. The white arrow indicates that the axillary buds of the lateral branches became flower buds in the dt1 mutant (e) compared with the young leaf formed in the wild type Shixiya 1 (d) (Scale bar = 2 cm)

Fig. 2

The long section analysis of the terminal buds. Microscopic observation of the terminal buds slices from one to five true leaf stages (from S1 to S5) in the dt1 mutant and the wild type Shixiya 1. LP stands for leaf primordium, SAM stands for shoot apical meristem and FM stands for floral meristem. Scale bar = 0.5 mm

Fig. 3

Fine mapping and cloning of determinate growth 1. a QTL analysis of the determinate growth phenotype in an F₂ segregating population, the determinate growth locus was mapped on chromosome A07 from 7.31 Mb to 24.57 Mb. The red arrow indicates the only window with a ΔSNP value exceeding the 99% significance threshold confidence interval across the whole genome. The ΔSNP index (SNP index of the determinate growth bulk population subtracted from that of the indeterminate growth bulk population) and its 99% confidence interval are shown as black curves and red dotted lines (ΔSNP index > 0.5), respectively. b Genetic mapping of the determinate growth locus in a population with 150 F₂ recessive individual plants by KASP. The number of recombinants was shown below the blue line. c The alternative 5′ splice site from GG to AG mutation in the first exon of Ga07G1189 resulted in a 31 amino acids (VYNGHEFFPSAVTNKPKVEVHGGDMRSFFTL) deletion in the putative protein sequence. d The G/GT splice site in TFL1 loci of other plants. In the gray dot box, the original G/GT splice site of GaTFL1 is marked in red and other G/GT sites are marked in orange

Fig. 4

Silencing of GaTFL1 results in terminal flower plants similar to dt1 phenotype. a The phenotype of Gossypium arboreum line DQJ after silencing endogenous GaTFL1 and blank vector infiltration plants. The red arrow indicates that the SAM became terminal flower in TRV: GaTFL1 plant compared with the young leaf formed in blank vector control TRV: 00 plant, and the white arrow indicates that the axillary buds of the lateral branches became flower buds in TRV: GaTFL1 plant compared with the young leaf formed in blank vector control TRV: 00 plant. b The level of GaTEL1 transcript in different tissues of GaTFL1-silenced (TRV: GaTFL1) plants and the negative control (TRV: 00). Two-tailed Student t-test was used for paired comparison of the GaTFL1 gene in TRV: GaTFL1 and TRV: 00 different tissues, respectively (**P < 0.01 or *P < 0.05)

Fig. 5

Transcriptomic comparison of the dt1 mutant versus Shixiya 1. a Venn diagram showing the overlaps between the different stages of the dt1 mutant and Shixiya 1. The number above each stage designation is the total transcripts detected in that stage(s). b Volcano plot of differentially expressed genes (DEGs) between the terminal bud of the dt1 mutant and Shixiya 1 at T₃ stage (flower budding stage, with visible flower buds). c The top 10 KEGG enriched pathways of up-regulated genes. d The top 10 KEGG enriched pathways of down-regulated genes. The top 10 KEGG enriched pathways are selected by p. adjust value sorting. Count, the bubble size, represents the number of enriched genes. GeneRatio represents the ratio of the number of DEGs corresponding to the pathway to the number of DEGs corresponding to the pathway database

Fig. 6

The protein structure and plant architecture analysis of the different types of TFL1 alleles. a The 3D protein structure of TFL1 and plant architecture of indeterminate growth cotton. The amino acids of the first exon is marked in gray, of which the 31 amino acids deletion of dt1 in the first exon is marked in light blue. b The 3D protein structure of Ghnb-1 to Ghnb-4 and plant architecture of Ghnb phenotype. The amino acids of the first exon is marked in gray, and the mutation of D73N amino acid is marked in purple. c The 3D protein structure of Gbnb-1 and plant architecture of Gbnb phenotype. The amino acids of the first exon is marked in gray, and the mutation of P113S amino acid is marked in bule. d The 3D protein structure of Gadt1 and plant architecture of Gadt1 phenotype. Amino (N) and carboxy (C) termini are labeled. White arrows in (a), (b) and (c) represent the missing β-sheets in (d). Green triangles represent monopodial SAMs, blue triangles represent sympodial SAMs, black arrows represent vegetative branches, and red circles represent bolls

Fig. 7

The hypothesis model of functional mechanism of TFL1 in plant architecture regulation. TFL1 associated complex are involved in the transport and translocation of sugar, key metabolites (e.g., flavonoids, toxin) or phytohormones (e.g., auxin) for the SAM tissue development. In these processes, TFL1 interacts with NOT2a or lipid di 18:1-PC to regulate the expression and function of genes encoding transporters responsible for auxin, sugar, flavonoids and toxin through epigenetic modification pathway or direct structure modification. With this mechanism, TFL1 function upstream of the auxin, sugar, flavonoids and toxin transport pathways to coordinate the vegetative and reproductive development and plant architecture. (Red circles around TFL1 represent lipid di 18:1-PC; Green triangles represent monopodial SAMs, blue triangles represent sympodial SAMs, black arrows represent vegetative branches, and red circles represent bolls)