Phenotypic analyses of rice lse2 and lse3 mutants that exhibit hyperaccumulation of starch in the leaf blades

Abstract

Background: To identify genes that potentially regulate the accumulation, mobilization, and transport of photoassimilates in rice (Oryza sativa L.) leaves, we recently screened a mutant collection of rice by iodine staining to visualize leaf starch contents. From this screening, we isolated a rice mutant that exhibits hyperaccumulation of starch in leaves and designated it as the Leaf Starch Excess 1 (LSE1) mutant. Here, we report two other rice LSE mutants, LSE2 and LSE3.

Results: Unlike lse1 plants, lse2 and lse3 plants displayed retarded growth; lse2 showed an extremely dwarf phenotype and rarely survived in paddy fields; lse3 showed inhibited growth with pale green leaf blades, low tiller numbers, reduced height, and low grain yield. In lse2 and lse3 plants, the mature source leaves contained larger amounts of starch and sucrose than those in wild-type and lse1 plants. Furthermore, microscopic observations of leaf transverse sections indicated that hyperaccumulation of starch in chloroplasts of mesophyll and bundle sheath cells occurred in lse2 and lse3 plants, while that in vascular cells was noticeable only in lse3 leaves.

Conclusions: The distinct phenotypes of these three LSE mutants suggest that the LSE2 and LSE3 mutations occur because of disruption of novel genes that might be involved in the path of sucrose transport from mesophyll cells to phloem sieve elements in rice leaves, the mechanism for which has not yet been elucidated.

Figure 1

Starch-excess phenotypes of rice lse mutants at the fifth leaf stage. Rice seedlings were sampled in the morning and subjected to iodine staining (A) or to microscopic observation (B–E). A, typical results of iodine staining in WT, lse1, lse2, and lse3 seedlings. Note that the youngest fifth leaf blades were still elongating very slowly and were not stained in lse2. Asterisks indicate the fifth leaf blade of rice seedlings. Bar = 10 cm. B–E, representative TEM images of transverse sections of fully-developed leaf blades of WT (B), lse1 (C), lse2 (D), and lse3 (E). The fourth leaf blades were used for lse2; fifth leaf blades were used for the others. Arrowheads indicate starch granules in mesophyll chloroplasts. Scale bars = 1 μm.

Figure 2

Microscopic observations of transverse sections of fully developed leaf blades of seedlings. The same tissue samples as shown in Figure 1B–E were used. A–D, light micrographs of periodic acid–Schiff-stained semithin sections. Dark red or purple dots showing accumulation of polysaccharides can be seen in lse1 (B), lse2 (C), and lse3 (D), and not in WT (A). Polysaccharide accumulation was pronounced in mesophyll cells (M) and was also evident in bundle sheath cells surrounding vascular bundles (black arrowheads). Blue arrowheads indicate polysaccharide accumulation within the large vascular bundle in lse3 (D). Scale bars = 100 μm. E–H, TEM of small or medium vascular bundles in wild type (E), lse1 (F), lse2 (G), and lse3 (H). Black arrowheads indicate starch granules in bundle sheath chloroplasts; blue arrowheads Indicate starch granules in vascular parenchyma cells. Scale bars = 2 μm. BS, bundle sheath cell; CC, companion cell; M, mesophyll; P, phloem; SE, sieve element; V, vessel element.

Figure 3

Two-month-old plants grown in pots under glasshouse conditions. Scale bar = 20 cm.

Figure 4

Comparison of concentrations of NSCs between WT and lse mutants. Leaf blades (LB) and sheaths (LS) of fifth-leaf-stage seedlings were sampled at the beginning of the light period (A,C) and at the beginning of the dark period (B,D). The fifth leaf blades and sheaths (5^th) were used for determination of starch, sucrose (Suc), glucose (Glc), and fructose (Fru). The fourth leaves (4^th) were also used in lse2. Values represent the averages of four plants ± SE. Differences between the WT and each lse mutant were analyzed by Student’s t-test. Significant differences are indicated by asterisks (*P ≤ 0.05; **P ≤ 0.01).

Figure 5

Comparison of concentrations of NSCs between WT and lse mutants grown under field conditions. Leaf blades, sheaths, and internodes were sampled between 9:00 and 10:00 AM at the heading stage. The flag (top) leaf blades (A) and sheath (C), the second-top leaf blades (B) and sheaths (D), the top (E) and second-top (F) internodes were used for determination of starch, sucrose (Suc), glucose (Glc), and fructose (Fru). Values represent the averages of four plants ± SE. Differences between the WT and each lse mutant were analyzed by Student’s t-test. Significant differences are indicated by asterisks (*P ≤ 0.05; **P ≤ 0.01).