The origin, initiation and development of axillary shoot meristems in Lotus japonicus

Abstract

Background and aims: Lotus japonicus 'Gifu' develops multiple axillary shoots in the cotyledonary node region throughout the growth of the plant. The origin, initiation and development of these axillary meristems were investigated.

Methods: Morphological, histological and mRNA in situ analyses were done to characterize the ontogeny of cotyledonary axillary shoot meristems in Lotus. Morphological characterization of a putative Lotus shoot branching mutant (super-accessory branches) sac, is presented.

Key results: By using expression of an L. japonicus STM-like gene as a marker for meristematic tissues, it was demonstrated that groups of cells maintained in the meristematic state at the cotyledonary axil region coincide with the sites where additional axillary meristems (accessory meristems) form. A Lotus shoot branching mutant, sac, is a putative Lotus branching mutant characterized by increased proliferation of accessory shoots in all leaf axils including the cotyledons.

Conclusion: In Lotus, axillary shoot meristems continually develop at the cotyledonary node region throughout the growth of the plant. These cotyledonary primary and accessory axillaries arise from the position of a meristematic zone of tissue at the cotyledonary node axil region.

Fig. 1

Diagram showing wild-type Lotus (A) and wild-type Arabidopsis (B) branching architecture. The primary shoot is indicated by a black arrow. The primary axillary buds/shoots are indicated by grey arrows. Accessory buds/shoots are indicated by blue arrows. Floral meristems are indicated by closed circles. Cotyledons are indicated by green ovals, and leaves by grey ovals. Numbers indicate the order of emergence of axillary and accessory buds.

Fig. 2

Shoot branching patterns in Lotus japonicus. (A) A 2-week-old Lotus seedling. (B) A 5-week-old plant. (C) A mature Lotus shoot. (D) Cotyledonary node of a 5-week-old plant; the cotyledon has been abscised. (E) A 3-week-old seedling. (F–H) A mature Lotus plant showing (F) node 1, (G) node 5, (H) node 6. ps, primary shoot; axb, primary axillary bud; axs, primary axillary shoot; acb, accessory bud; acs, accessory shoot; cn, cotyledonary node; cot, cotyledon; fb, flower branch.

Fig. 3

Number of axillary buds/shoots at the cotyledonary node and average height of Lotus throughout 77 d growth duration.

Fig. 4

SEM images showing (A) a newly detached cotyledon and (B) a detached cotyledon after growth in culture medium for 2 months. New accessory buds are indicated by arrows. axb, primary axillary bud; axs, primary axillary shoot; acb, accessory bud. Scale bars in (A) and (B) are 400 μm.

Fig. 5

Longitudinal sections of (A) mature embryo, (B) newly imbibed seed, (C) 4-day-old seedling and (D) 10-day-old seedling, showing densely staining meristematic tissues. (E) SEM of a 7-day-old seedling Arrows indicate the relative positions of transverse sections shown in (F) and (G). (H) A transverse section through the cotyledonary node of a more mature seedling. sam, shoot apical meristem; cot, cotyledon; axm, axillary meristem; axb, primary axillary bud; axs, primary axillary shoot; acm, accessory meristem; acb, accessory bud; ps, primary shoot, mt, meristematic tissues. Scale bars in A–G, 200 μm; H, 500 μm.

Fig. 6

(A) Comparison of the deduced amino acid sequence for the LjSTM-like gene (Lj S53343) with that of STM (Long et al., 1996) and the Medicago knotted 1-like gene (Koltai et al., 2001). Vector NTI was used to make the alignment and the deduced distance tree. Identical residues are darkly shaded. Similar residues are lighly shaded. Dashed lines indicate gaps introduced by the programme to attain maximum alignment. (B) Distance tree of different STM cDNAs.

Fig. 7

LjSTM-like mRNA (indicated by dark staining) accumulates in shoot apical meristems of Lotus japonicus. Longitudinal sections of (A) late heart/early torpedo, and (B) bent stage embryos showing LjSTM-like transcript accumulation in the presumptive shoot apical meristems (arrows). (C) A transverse section of a young Lotus seedling showing LjSTM-like transcript accumulation in the SAM (dark stained group of cells indicated by an arrow). P1 and P2 leaf primordia curve around the SAM. Longitudinal sections of (D) a 4-day-old seedling showing transcript accumulation in the axillary meristems. (E) In a 7-day-old seedling, transcript accumulation is restricted to the shoot apical meristem of an axillary bud. cot, cotyledon; ps, primary shoot; axm, axillary meristem; axb, axillary bud; P1 and P2, leaf primordia where P2 is older than P1. Scale bars in (A–C) 200 μm; (D) 100 μm; and (E) 200 μm.

Fig. 8

Localization of LjSTM-like mRNA (indicated by dark staining) in Lotus. (A) A 2-day-old seedling. A plane and a green line indicate the positions of a sagittal longitudinal section as shown in (B) and a transverse section as shown in (C), respectively. In situ hybridization shows that LjSTM-like transcripts accumulate in the cotyledon axils of a 7-day-old seedling as shown in adjacent median longitudinal sections (D) and (E). As the bud develops, LjSTM-like transcript accumulation is restricted to the shoot apical meristem of the new axillary bud as shown in (E). (F) Longitudinal section of a 2-day-old seedling hybridized with a sense probe. sam, shoot apical meristem; ps, primary shoot; axm, primary axillary meristem; axb, primary axillary bud; axs, primary axillary shoot; acb, accessory bud; acs, accessory shoot; cot, cotyledon; mt, meristematic tissues). Scale bars in (B–D) are 200 μm and that in (E) is 100 μm.

Fig. 9

Phenotype of the Lotus super-accessory branching (sac) mutant. Mature (A) wild-type, (B) sac, 77 d after planting (DAP. Cotyledonary nodes of (C) wild type and (D) sac, 2 months after planting. (E–H) Node 1 of Lotus plants, wild type at (E) 35 DAP and (F) 77 DAP, sac at (G) 35 DAP and (H) 77 DAP. Numerous accessory shoots develop in node 1 of sac1 (G) at 77 DAP, and accessory shoots are not distinct from the primary axillary shoot, hence these buds/shoots were not labelled. ps, primary shoot; axs, primary axillary shoot; acb, accessory bud; numbers 1–6 refer to accessory buds according to the order of emergence.