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. 2024 Mar 5:15:1363182.
doi: 10.3389/fpls.2024.1363182. eCollection 2024.

Mutating alfalfa COUMARATE 3-HYDROXYLASE using multiplex CRISPR/Cas9 leads to reduced lignin deposition and improved forage quality

Tezera W Wolabu  1 , Kashif Mahmood  1 , Fang Chen  2 , Ivone Torres-Jerez  1 , Michael Udvardi  3 , Million Tadege  1 , Lili Cong  4 , Zengyu Wang  4 , Jiangqi Wen  1
Affiliations

Mutating alfalfa COUMARATE 3-HYDROXYLASE using multiplex CRISPR/Cas9 leads to reduced lignin deposition and improved forage quality

Tezera W Wolabu et al. Front Plant Sci. .

Abstract

Alfalfa (Medicago sativa L.) forage quality is adversely affected by lignin deposition in cell walls at advanced maturity stages. Reducing lignin content through RNA interference or antisense approaches has been shown to improve alfalfa forage quality and digestibility. We employed a multiplex CRISPR/Cas9-mediated gene-editing system to reduce lignin content and alter lignin composition in alfalfa by targeting the COUMARATE 3-HYDROXYLASE (MsC3H) gene, which encodes a key enzyme in lignin biosynthesis. Four guide RNAs (gRNAs) targeting the first exon of MsC3H were designed and clustered into a tRNA-gRNA polycistronic system and introduced into tetraploid alfalfa via Agrobacterium-mediated transformation. Out of 130 transgenic lines, at least 73 lines were confirmed to contain gene-editing events in one or more alleles of MsC3H. Fifty-five lines were selected for lignin content/composition analysis. Amongst these lines, three independent tetra-allelic homozygous lines (Msc3h-013, Msc3h-121, and Msc3h-158) with different mutation events in MsC3H were characterized in detail. Homozygous mutation of MsC3H in these three lines significantly reduced the lignin content and altered lignin composition in stems. Moreover, these lines had significantly lower levels of acid detergent fiber and neutral detergent fiber as well as higher levels of total digestible nutrients, relative feed values, and in vitro true dry matter digestibility. Taken together, these results showed that CRISPR/Cas9-mediated editing of MsC3H successfully reduced shoot lignin content, improved digestibility, and nutritional values without sacrificing plant growth and biomass yield. These lines could be used in alfalfa breeding programs to generate elite transgene-free alfalfa cultivars with reduced lignin and improved forage quality.

Keywords: CRISPR/Cas9; MsC3H; alfalfa; digestibility; forage quality; gene editing; lignin composition; nutritional value.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Schematic illustration of alfalfa COUMARATE 3-HYDROXYLASE (MsC3H) gene structure with designed multiplex gRNAs-CRISPR/Cas9 vector and genome editing efficiency in alfalfa. (A) MsC3H gene structure and four guide RNA sites with specific sequences in the coding region. (B) Mutation efficiency (%) of four guide RNAs at different target sites of MsC3H. (C) Illustration of the multiplex construct of MsC3H-gRNA1,2,3,4-CRISPR/Cas9 vector.
Figure 2
Figure 2
Analysis of lignin content and composition in stem tissues of three selected Msc3h mutant lines Msc3h-013, Msc3h-121 and Msc3h-158. (A) S lignin content (%) in stem tissues of Msc3h lines and EV. (B) G lignin content (%) in stem tissues of Msc3h lines and EV. (C) H lignin content (%) in stem tissues of Msc3h lines and EV. (D) Total (H+G+S) lignin content (%) in stem tissues of Msc3h lines and EV. (E) S/G ratio in stem tissues of Msc3h lines and EV. (F) Phenotype of mutant lines Msc3h-013, Msc3h-121, and Msc3h-158 with EV at early flower bud vegetative growth stage. Data represent mean values (± SD; n = 6). Statistics was analyzed using Student’s t-test (* p < 0.05; ** p < 0.01).
Figure 3
Figure 3
Molecular analysis of MsC3H mutation events (deletions/insertion/substitution) generated by multiplex gRNAs-CRISPR/Cas9 gene editing at four target sites of MsC3H. Mutation events (deletions, insertions, substitutions) of mutant lines Msc3h-013, Msc3h-121, and Msc3h-158 occurred at each specific target site (gRNA1, gRNA2, gRNA3 and gRNA4) compared to wild type (WT). Deletions are indicated by red dashed lines; insertions or substitutions are indicated by red letters. PAMs are indicated by red underlined italicized letters. The four allelic copies are designated as red allele-1 (A1), allele-2 (A2), allele-3 (A3), and allele-4 (A4).
Figure 4
Figure 4
Analysis of forage quality in aboveground (leaves and stem) tissues of Msc3h mutant lines. (A) Acid Detergent Fiber (ADF) content (%) in Msc3h lines and EV. (B) Neutral Detergent Fiber (NDF) content (%) in Msc3h lines and EV. (C) Total Digestible Nutrients (TDN) content (%) in Msc3h lines and EV. (D) Relative Feed Value (RFV) (%) in Msc3h lines and EV. (E) In Vitro True Dry Matter Digestibility (IVTDMD) (%) in Msc3h lines and EV. (F) Crude protein content (%) in Msc3h lines and EV. Data represent mean values (± SD; n = 6) and were analyzed statistically using Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 5
Figure 5
Analysis of forage macro-minerals contents in aboveground (leaves and stem) tissues of mutant lines Msc3h-013, Msc3h-121 and Msc3h-158 and EV. (A) Phosphorus (P) content (%). (B) Calcium (Ca) content (%). (C) Potassium (K) content (%). (D) Magnesium (Mg) content (%). Data represent mean values (± SD; n = 6) and were analyzed statistically using Student’s t-test (* p < 0.05; ** p < 0.01).
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