Thaumatin-like Gene TLP1b Confers to Seed Oil Content and Resistance to Sclerotinia sclerotiorum in Arabidopsis

Abstract

The synergistic optimization of yield and abiotic/biotic resistance is of great significance in plant breeding. However, the genomic mechanisms underlying the selection for environmental adaptation and yield-related traits remain poorly understood. In this study, we identified a thaumatin-like protein (TLP), AtTLP1b, which was shown to pleiotropically regulate seed oil content and resistance to Sclerotinia sclerotiorum by gene knockout and overexpressing experiments in Arabidopsis. The oil composition oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3) and eicosenoic acid (C20:1) were altered significantly in overexpressing and knockout lines. RNA-seq analysis revealed that overexpression of AtTLP1b significantly downregulated the expression levels of genes involved in wax, suberin synthesis, oxylipin metabolism and plant-pathogen interaction. Furthermore, more than half of the genes involved in the circadian rhythm-plant pathway were differentially expressed in the overexpressing lines. We propose that AtTLP1b primarily inhibits fatty acid synthesis and plant immune responses via the circadian rhythm-plant pathway. Our findings suggest that AtTLP1b plays a vital role in simultaneous improvement of seed oil content and resistance to S. sclerotiorum and provides a valuable resource for molecular breeding.

Keywords: Arabidopsis; AtTLPb; S. sclerotiorum; circadian rhythm; seed oil content.

Figure 1

AtTLP1b is a typical TLP localized at the ER. (A) Schematic diagram of AtTLP1b protein structure. The red and orange boxes represent the signal peptide and the thaumatin domain of AtTLP1b.The black boxes represent protein without structural features. The green brackets represent the disulfide bonds. (B) Phylogenetic tree analysis of the TLP family in Arabidopsis was conducted using 13 amino acid sequences. The evolutionary distances were calculated, and the optimal tree with a total branch length of 3.40385098 was constructed. The tree is scaled, with branch lengths reflecting the evolutionary distances used for phylogenetic inference. Gaps and missing data were excluded from the analysis, resulting in a final dataset comprising 207 aligned positions. (C) From left to right, GFP represents green fluorescence under confocal microscopy, and ER-marker was used to visualize ER, which exhibited red fluorescence under confocal microscopy. Merged indicates the overlap of GFP channel and ER-marker channel, showing yellow fluorescence under confocal microscope. Scale bars = 10 μm.

Figure 2

Gene expression analysis and the seed oil content of the overexpressing and knockout lines of AtTLP1b in A. thaliana. (A) Relative gene expression of the overexpressing lines. Three technical duplicates were used for RT-qPCR analysis. (B) The selection of sgRNA for the knockout vector and the editing types of T₁ and T₂ generations of knockout lines. The gray box represents 5′UTR or 3′UTR, the black box represents exons, and the yellow box represents thaumatin domain, and the black straight line represents intron. The blue triangle represents two sgRNA target sites, the red text represents the sgRNA target sequence, and the underlined text is the PAM sequence “NGG”. (C) The seed oil content of overexpressing lines and knockout lines of AtTLP1b. Three biological repetitions were used for analysis. (D) The content of each fatty acid component. The asterisk represented significant differences between AtTLP1b overexpressing lines/knockout lines and Col-0 (* p < 0.01, ** p < 0.01, *** p < 0.001. Student’s t-test).

Figure 3

Overexpression and knockout of AtTLP1b affect the resistance to S. sclerotiorum in Arabidopsis. (A) The overexpressing lines and knockout lines of AtTLP1b along with Col-0 were challenged with S. sclerotiorum strain 1980. Scale bars = 1 cm. Photographs were taken at 24 hpi. (B) Statistical analysis of the lesion area induced by 1980 on overexpressing lines and knockout lines of AtTLP1b together with Col-0 at 24 hpi. Each small red/black dot represented a measured value, the green dotted line represented the median, and the asterisk represented significant differences between AtTLP1b overexpressing lines/knockout lines and Col-0 (*** p < 0.001. Student’s t-test).

Figure 4

Comparison of gene numbers and pathways involved in acyl-lipid metabolism of siliques at four different developmental stages. (A) The proportions of identified ALM genes to the total number DEGs of each developmental stage. Total represents the total number of DEG genes. (B) Percentage of genes involved in the 16 pathways of ALM in each developmental stage.

Figure 5

KEGG pathway enrichment results of the overlapping DEGs at four silique development stages of two overexpressing lines versus Col-0. Circle, square, triangular and inverted triangle represent the KEGG pathways enriched by DEGs of 16DAF, 12DAF, 8DAF and 4DAF silique development stages, respectively. The larger the shape, the more genes are enriched in this pathway. The higher the −logQvalue, the higher the significance of the pathway.

Figure 6

The expression of the overlapping DEGs of two overexpressing lines in circadian rhythm-plant pathway. The red box and green box in the pathway represent that the gene is up-regulated and down-regulated compared to Col-0 at least one developmental stage of siliques. The four squares from left to right in the heatmap represent the silique samples of 4 DAF, 8 DAF, 12 DAF, and 16 DAF, respectively. The four squares at the top of the heatmap belong to the OE-9 line, while the four squares at the bottom belong to the OE-18 line. The solid arrows represent direct effect, and the dotted arrows represent indirect effect.

Figure 7

Working model of AtTLP1b affects seed oil content and resistance to S. sclerotiorum in Arabidopsis. The solid arrows represent direct effect, and the dashed arrow represents possible indirect effect.