Knockdown of the symbiotic sucrose synthase MtSucS1 affects arbuscule maturation and maintenance in mycorrhizal roots of Medicago truncatula

Abstract

The relevance of the symbiosis-induced Medicago truncatula sucrose synthase gene MtSucS1 for an efficient arbuscular mycorrhiza (AM) was studied using two independent antisense lines that displayed up to 10-fold reduced SucS1 levels in roots. Mycorrhizal MtSucS1-reduced lines exhibited an overall stunted aboveground growth under inorganic phosphorus limitation. Apart from a reduced plant height, shoot weight, and leaf development, a delayed flowering, resulting in a lower seed yield, was observed. In addition, the root-to-shoot and root weight ratios increased significantly. Gene expression studies demonstrated a major reversion of AM-associated transcription, exhibiting a significant repression of well-known plant AM marker and mycosymbiont genes, together indicating a diminished AM fungus colonization of MtSucS1-antisense lines. Concomitantly, gas chromatography-mass spectrometry-based metabolite profiling revealed that mycorrhizal MtSucS1-reduced lines were affected in important nodes of the carbon, nitrogen, and phosphorus metabolism, accentuating a physiological significance of MtSucS1 for AM. In fact, antisensing MtSucS1 provoked an impaired fungal colonization within the less abundant infected regions, evident from strongly reduced frequencies of internal hyphae, vesicles, and arbuscules. Moreover, arbuscules were early senescing, accompanied with a reduced development of mature arbuscules. This defective mycorrhiza status correlated with reduced phosphorus and nitrogen levels and was proportional to the extent of MtSucS1 knockdown. Together, our results point to an important role for MtSucS1 in the establishment and maintenance of arbuscules in the AM symbiosis.

Figure 1.

Phenotypes of P_i-limited MtSucS1-as line as12 and controls colonized by G. mosseae. Phenotypes of mycorrhizal MtSucS1-as12 and corresponding mycorrhizal control lines are shown at 32 dpi (multiple plants grown in parallel; A), 32 dpi (single plant; B), 37 dpi (single plant; C), 50 dpi (multiple plants grown in parallel; D), 50 dpi (single plant; E), and 57 dpi (single plant; F). Order of plant lines, starting from the left position, is MtSucS1-as12 and control. Plant material was harvested at 50 dpi.

Figure 2.

Growth and developmental characteristics of MtSucS1-as lines. A, Leaf number development in mycorrhizal MtSucS1-as and control lines. B, Relative vegetative and reproductive characteristics of mycorrhizal MtSucS1-as and control lines at 50 dpi (n = 30 individuals per line). DW, Dry weight; FW, fresh weight; RWR, root weight ratio as DW_root per DW_plant. C, Specific root parameters of mycorrhizal MtSucS1-as and control lines. The specific root length, surface area, root volume, average root diameter, and root tip number of mycorrhizal MtSucS1-as and control lines were expressed as root parameter per unit of shoot dry weight (DW_shoot) at 50 dpi (n_as = 9, n_c = 16 mycorrhizal plants). D, Root and shoot C, N, and P contents of mycorrhizal MtSucS1-as and control lines. The mineral contents were calculated per unit dry weight in 50 dpi-old mycorrhizae (n = 8 biological replicates per line). Significantly different means ± se are indicated as follows: ^a 0.05 > P ≥ 0.01, ^b P < 0.01. Relative values are in comparison with control levels set to 100%. Gray bars, AM MtSucS1-as line as12; dark gray bars, AM MtSucS1-as line as19; white bars, AM control lines; gray squares and dashed gray line, NM MtSucS1-as; white circles and dashed black line, NM controls. Yield indicates number of pods at day of harvest. cc, Construction cost; % c, Percentage in relation to controls.

Figure 3.

Relative gene expression in mycorrhizal MtSucS1-as compared with control lines. Relative gene expression in lines as19 and as12 is given as mean MtSucS1-as/control ratios. Relative expression values were first calculated from MtTef1α-normalized values and were further normalized to the arbuscule activity-specific transcriptional coefficient MtPT4/GmTef1. Tentative consensus sequences are sorted via x-fold changes with respect to line as19. A to C, Relative quantities of mycorrhizal M. truncatula RNA per unit of MtPT4/GmTef1. D, Relative quantities of G. mosseae RNAs per unit of MtPT4/GmTef. Measurements are based upon five biological replicates. Gray bars, Mycorrhizal MtSucS1-as12; dark gray bars, mycorrhizal MtSucS1-as19. Gm, G. mosseae; TC, tentative consensus sequence.

Figure 4.

Mycorrhiza phenotypes of MtSucS1-as and control lines. A, G. mosseae root length colonization and specific internal colonization of intraradical hyphae, arbuscules, and vesicles in mycorrhizal regions at 50 dpi. sRC, Specific root colonization. B, Root length colonization time course of mycorrhizal MtSucS1-as19 and control lines as revealed from sequentially harvested plants. Each point represents the mean ± se of at least three plants. Regression curves for mycorrhizal lines are shown. C to K, Representative fluorescence images of AlexaFluor488-stained mycorrhizal root segments at 50 dpi. C to E, MtSucS1-as12 (C), MtSucS1-as19 (D), and control lines (E) depicting the significantly reduced mycorrhiza status in both MtSucS1-as lines. Bars = 100 μm. F to K, Pronounced arbuscule senescence in MtSucS1-as12 and -as19 segments displayed by high proportions of degenerating (dgA) and dead (deA) arbuscules. Bars = 75 μm (F–H) and 30 μm (I–K). L, Distribution of the arbuscule developmental stages in MtSucS1-as lines at 50 dpi compared with controls set to 100%. Significantly different means ± se are indicated as follows: ^a 0.05 > P ≥ 0.01, ^b P < 0.01. Gray bars, MtSucS1-as line as12; dark gray bars, MtSucS1-as line as19; white bars, controls; gray squares and dashed gray line, mycorrhizal MtSucS1-as19; white circles and solid line, controls. Relevant morphological fungal structures such as intraradical hyphae (H), arbuscules (A), and septae (S) are highlighted with arrowheads. AC, Arbuscule colonization; HC, intraradical hyphae; mA, mature arbuscule; % c, percentage in relation to controls; VC, vesicle.