From model to crop: functional characterization of SPL8 in M. truncatula led to genetic improvement of biomass yield and abiotic stress tolerance in alfalfa

Abstract

Biomass yield, salt tolerance and drought tolerance are important targets for alfalfa (Medicago sativa L.) improvement. Medicago truncatula has been developed into a model plant for alfalfa and other legumes. By screening a Tnt1 retrotransposon-tagged M. truncatula mutant population, we identified three mutants with enhanced branching. Branch development determines shoot architecture which affects important plant functions such as light acquisition, resource use and ultimately impacts biomass production. Molecular analyses revealed that the mutations were caused by Tnt1 insertions in the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 8 (SPL8) gene. The M. truncatula spl8 mutants had increased biomass yield, while overexpression of SPL8 in M. truncatula suppressed branching and reduced biomass yield. Scanning electron microscopy (SEM) analysis showed that SPL8 inhibited branching by directly suppressing axillary bud formation. Based on the M. truncatula SPL8 sequence, alfalfa SPL8 (MsSPL8) was cloned and transgenic alfalfa plants were produced. MsSPL8 down-regulated or up-regulated alfalfa plants exhibited similar phenotypes to the M. truncatula mutants or overexpression lines, respectively. Specifically, the MsSPL8 down-regulated alfalfa plants showed up to 43% increase in biomass yield in the first harvest. The impact was even more prominent in the second harvest, with up to 86% increase in biomass production compared to the control. Furthermore, down-regulation of MsSPL8 led to enhanced salt and drought tolerance in transgenic alfalfa. Results from this research offer a valuable approach to simultaneously improve biomass production and abiotic stress tolerance in legumes.

Keywords: Medicago sativa; Medicago truncatula; alfalfa; biomass yield; branching; drought tolerance; forage legume; salt tolerance.

Figure 1

Phenotype and biomass yield of three Tnt1 mutants of SPL8 in M. truncatula. (a) Phenotype of 4‐week‐old mutant plants. (b) Eight‐week‐old plants of mutants and control. (c) Branch development in the control and spl8 mutants. (d) Tnt1 insertion sites in SPL8 of the three mutants. (e) Semiquantitative PCR indicates that MtSPL8 expression is abolished in spl8 mutants. (f) Fresh and dry biomass yields of mutant and control plants. Values represent mean ± S.D. of five biological replicates and are analyzed by Student's t‐test (**P < 0.01).

Figure 2

Effects of SPL8 overexpression in M. truncatula. (a) Mature plants of control and SPL8 overexpression transgenic lines (SPL8OE). (b) SPL8 relative expression levels in transgenic plants. (c) Branch density (primary branches produced in one centimeter of main stem) of overexpression lines and control. (d) Fresh biomass yield of overexpression lines and control. (e) Dry biomass yield of overexpression lines and control. Values represent means ± S.D. of three biological replicates and are analyzed by Student's t‐test (*P < 0.05, ***P < 0.001).

Figure 3

SAM development in spl8 mutant (spl8‐2), control and overexpression plant (SPL8OE‐18) in M. truncatula. (a) SAM structure in the control. (b) SAM structure in spl8. (c) SAM structure in SPL8OE with complete removal of all surrounding tissue and leaflets. AB, axillary bud; ILP, incipient leaf primordia; LL, lateral leaflet primordia; TL, terminal leaflet primordia; ST, stipule primordia.

Figure 4

Genetic modification of MsSPL8 significantly altered shoot architecture and biomass yield in alfalfa (Medicago sativa). (a) Two‐month‐old plants of control, MsSPL8 down‐regulation (MsSPL8Ri‐14) and overexpression transgenics (MsSPL8OE‐23). (b) Relative expression levels of MsSPL8 in the MsSPL8Ri, control and MsSPL8OE plants. (c) Branch density (the total branch numbers produced in one centimeter of main stem) of the MsSPL8Ri, control and MsSPL8OE plants. (d) Fresh and dry biomass yields (gram) of the MsSPL8Ri, control and MsSPL8OE plants. Values represent means ± S.D. of six biological replicates and are analyzed by Student's t‐test (***P < 0.001).

Figure 5

Down‐regulation of MsSPL8 markedly accelerated regrowth in alfalfa. (a) New shoots produced after cutting from the control and MsSPL8 down‐regulation transgenic plants (MsSPL8Ri). (b) Forage biomass (g) of the second harvest. (c) Percentage increase in biomass yield of the first and second harvests. Biomass of the second harvest is significantly higher than the first harvest in the transgenics. Values represent means ± S.D. of three biological replicates and are analyzed by Student's t‐test (**P < 0.01, ***P < 0.001).

Figure 6

Effects of salt and drought stress on transgenic alfalfa plants down‐regulated with MsSPL8 (MsSPL8Ri) or up‐regulated with MsSPL8 (MsSPL8OE). (a) Plants before salt treatment. (b) Phenotype after 2‐week salt treatment. (c) Phenotype after 3‐week salt treatment. (d) Plants before drought treatment. (e) Phenotype after 2‐week drought treatment (no watering). (f) Six days of recovery after re‐watering from 2‐week drought treatment. Experiments are conducted with four biological replicates.