LAX and SPA: major regulators of shoot branching in rice

Abstract

The aerial architecture of plants is determined primarily by the pattern of shoot branching. Although shoot apical meristem initiation during embryogenesis has been extensively studied by molecular genetic approaches using Arabidopsis, little is known about the genetic mechanisms controlling axillary meristem initiation, mainly because of the insufficient number of mutants that specifically alter it. We identified the LAX PANICLE (LAX) and SMALL PANICLE (SPA) genes as the main regulators of axillary meristem formation in rice. LAX encodes a basic helix-loop-helix transcription factor and is expressed in the boundary between the shoot apical meristem and the region of new meristem formation. This pattern of LAX expression was repeatedly observed in every axillary meristem, consistent with our observation that LAX is involved in the formation of all types of axillary meristems throughout the ontogeny of a rice plant. Ectopic LAX expression in rice caused pleiotropic effects, including dwarfing, an altered pattern of stem elongation, darker color, bending of the lamina joint, absence of the midribs of leaves, and severe sterility.

Fig. 1.

Phenotypic analysis of lax mutant. (A) A schematic of a rice plant (Left) and a panicle (Right). A rice plant is composed of a main stem called a main culm (mc) and a multiple tillers (t) produced as axillary buds. A panicle has several primary panicle branches (pb). A few secondary panicle branches (sb) and several lateral spikelets (ls) are produced on each primary panicle branch. Finally, the SAM of each panicle branch is transformed into a terminal spikelet (ts). (B) Panicle morphology of the lax mutant. WT (Left), lax-1 (Center), and lax-2 (Right) panicles. All lateral spikelets are absent, but fertile terminal spikelets are produced in lax-1, whereas formation of panicle branches and spikelets is severely reduced in lax-2.(C and D) OSH1 expression in an immature panicle at the secondary panicle branch initiation stage of WT (C) and lax-2 (D). Note: In a rice panicle, the bract leaves subtending branch shoots including panicle branches and lateral spikelets, are rudimentary and do not grow big. Red arrow shows the down-regulation of OSH1 in the site where a new meristem initiation. Green arrow shows a new axillary meristem. t, tiller; mc, main culm. (Bars = 50 μm.)

Fig. 3.

Positional cloning of the LAX gene. (A) Schematics of positional cloning. Position of the LAX locus was delimited within the 82-kb region straddling two P1 artificial chromosome clones, P0446G04 and P0460C04. Subsequently, the existence of a long deletion, which contains five predicted genes, PG1 to PG5, was identified in the lax-2 allele. (B) Lesions in lax alleles. An insertion of a retrotransposon was detected in the lax-1 allele. The sequence derived from the retrotransposon is shown in italics. A 59-bp region was deleted in lax-3. Amino acid substitutions were found in lax-4 and lax-5.(C) Amino acid sequence of LAX deduced from the cDNA sequence. The bHLH domain is shown in green. (D) Comparison of the bHLH domain of LAX and the most closely related gene of Arabidopsis (acc.At5g01310), PIF3 (AF100166), SPATULA (AF319540), maize B-Peru (S16594), and human cMYC (P23583).

Fig. 2.

Phenotypic analysis of spa and lax spa double mutants. (A) Comparison of the panicle phenotypes of the lax, spa single mutants, and the lax spa double mutant. Arrowheads indicate panicle nodes. (B–D) Close-up views of panicles in WT (B), lax-1 (C), and spa (D) mutants. (E) Effects of lax and spa mutations on the number of tillers, vegetative stage branching. Number of panicles, except for one on a main culm, are shown as the number of tillers. Number of plants examined were 68, 42, 136, and 6 for WT, lax-1, spa, and lax-1 spa, respectively. (F and G) Tiller buds of vegetative stage plants. Arrows indicate tiller buds in the WT plant (F). Tiller buds were not observed in lax spa double mutant plants (G).

Fig. 4.

Expression pattern of the LAX gene. RNA in situ hybridization of LAX (A–D, F–H, and J) and OSH1 (E, I, and K). (A) Longitudinal section of a vegetative SAM. (B–F) Longitudinal section of an inflorescence meristem at the primary branch differentiation stage (B), the secondary branch differentiation stage (C), and the spikelet initiation stage (D–F). LAX mRNA was specifically expressed in the upper-boundary layer between the meristem and the region in which the axillary meristem will develop. Comparison of the expression pattern of LAX (D) and OSH1 (E) in consecutive sections shows that LAX expression has been initiated where OSH1 down-regulation took place (red arrow). When the new axillary meristem started to grow, both LAX and OSH1 were expressed (green arrows). (F) The same expression pattern of LAX was repeatedly observed in the places where new axillary meristems initiate. (G) LAX mRNA was expressed in the tiller primordium. (H and I) LAX and OSH1 mRNA accumulation during tiller bud initiation. Analysis of consecutive sections indicated that LAX mRNA localization (H) is in two cell layers in the upper region of OSH1 expression (I). (J and K) LAX and OSH1 expressions in the late globular-stage embryo. OSH1 expression was observed in the region where a primary SAM initiates (arrowhead), whereas the signal of LAX expression was not observed. (Bars = 50 μm.)

Fig. 5.

Phenotypes of Act::LAX transgenic rice plants. (A) Ectopic expression of LAX in leaves of Act::LAX transgenic plants. The + indicates severity of the phenotype. (B) A representative Act::LAX transgenic rice plant (Left) and a WT plant (Right). (C) Lamina joint in an Act::LAX plant (Left) and a WT plant (Right). (D) Transverse section of leaf blades in an Act::LAX plant (Left) and WT plant (Right). The development of a midrib was observed in the WT leaf but not in the Act::LAX leaf. (E) Pollen grains in an Act::LAX transgenic plant (Left) and a WT plant (Right). Pollen in the transgenic plants look empty and shrunken. lg, ligule; lb, leaf blade; ls, leaf sheath; a, auricle; lj, lamina joint.