KNAT6: an Arabidopsis homeobox gene involved in meristem activity and organ separation

Abstract

The homeobox gene family plays a crucial role during the development of multicellular organisms. The KNOTTED-like genes from Arabidopsis thaliana (KNAT6 and KNAT2) are close relatives of the meristematic genes SHOOT MERISTEMLESS (STM) and BREVIPEDICELLUS, but their function is not currently known. To investigate their role, we identified null alleles of KNAT6 and KNAT2. We demonstrate that KNAT6 contributes redundantly with STM to the maintenance of the shoot apical meristem (SAM) and organ separation. Consistent with this role, the expression domain of KNAT6 in the SAM marks the boundaries between the SAM and cotyledons. The lack of meristematic activity in the knat6 stm-2 double mutant and the fusion of cotyledons were linked to the modulation of CUP-SHAPED COTYLEDON (CUC) activity. During embryogenesis, KNAT6 is expressed later than STM and CUC. In agreement with this fact, CUC1 and CUC2 were redundantly required for KNAT6 expression. These data provide the basis for a model in which KNAT6 contributes to SAM maintenance and boundary establishment in the embryo via the STM/CUC pathway. KNAT2, although the closest related member of the family to KNAT6, did not have such a function.

Figure 1.

KNAT6 Is Expressed in Boundaries. (A) Expression of pKNAT6-GUS was detected at the torpedo stage. The embryos were cleared. (B) Longitudinal section through a globular embryo. The KNAT6 mRNA was not detected. (C) Longitudinal section through a triangular embryo showing KNAT6 mRNA accumulation in the presumptive SAM. (D) and (E) Longitudinal sections through late-torpedo (D) and mature (E) embryos showing KNAT6 mRNA accumulation in the boundaries between the SAM and the cotyledons. (F) Transverse section through a mature embryo showing KNAT6 mRNA accumulation in a region surrounding the SAM. This region corresponds to the boundaries between the SAM (S) and the cotyledons (C) and the boundaries between the cotyledons. Bars = 50 μm.

Figure 2.

Characterization of the Loss-of-Function knat6 and knat2 Mutant Alleles. (A) RT-PCR analysis using KNAT6 gene-specific primers. After 38 cycles, the PCR products were transferred to a nylon membrane and hybridized with a ³³P-labeled KNAT6 probe. Amplification occurred in wild-type seedlings (lane 1) but not in knat6-1 and knat6-2 alleles (lanes 2 and 3, respectively), indicating that both alleles are null. Control amplification of APT1 indicates that equal amounts of cDNA were present in each sample. (B) RT-PCR analysis using KNAT2 gene-specific primers. After 40 cycles, the PCR products were transferred to a nylon membrane and hybridized with a ³³P-labeled KNAT2 probe. Amplification occurred in wild-type seedlings (lane 1) but not in the knat2-5 allele (lane 2), indicating that the knat2-5 allele is null. Control amplification of APT1 indicates that equal amounts of cDNA were present in each sample.

Figure 3.

Phenotype of the knat6 stm Double Mutant. All seedlings shown are 8 d old. Bars = 100 μm. (A) Wild-type seedling. The SAM gives rise to leaf primordia. (B) stm-2 seedling. (C) knat6-1 stm-2 seedling showing a fusion that extends to the blade of the cotyledons. (D) Longitudinal section through a wild-type vegetative meristem. (E) Longitudinal section through a stm-2 seedling. A primordium is visible between the cotyledons. (F) Longitudinal section through a knat6-1 stm-2 seedling. The cells are large and vacuolated.

Figure 4.

WET368 GUS Expression in knat6-1 Single Mutant and knat6-1 stm-2 Double Mutant. (A) knat6-1/knat6-1 mature embryo showing GUS activity in the SAM boundaries. This activity was identical in the wild-type embryo. (B) knat6-1/knat6-1 stm-2/stm-2 mature embryo showing a dramatic reduction of GUS activity. (C) knat6-1/knat6-1 8-d-old seedling showing GUS activity in the meristem. Seedlings were cleared. (D) stm-2/stm-2 8-d-old seedling exhibiting GUS activity. (E) knat6-1/knat6-1 stm-2/stm-2 8-d-old seedling exhibiting no GUS activity. Bars = 50 μm.

Figure 5.

Triple Mutants of knat6-1 stm-2 cuc1-1, knat6-1 stm-2 cuc2, and knat6-1 stm-2 cuc3-2. Eight-day-old seedlings of the wild type ([A] and [E]), cuc1-1 cuc2 ([B] and [F]), cuc1-1 cuc3-2 ([C] and [G]), cuc2 cuc3-2 ([D] and [H]), knat6-1 stm-2 ([I] and [M]), knat6-1 stm-2 cuc1-1 ([J] and [N]), knat6-1 stm-2 cuc2 ([K] and [O]), and knat6-1 stm-2 cuc3-2 ([L] and [P]).

Figure 6.

pKNAT6-GUS Expression in Mutant Backgrounds. (A) Wild-type mature embryo showing GUS activity in the SAM boundaries and in the tips of cotyledons. The embryo was cleared. (B) cuc1-1/cuc1-1 cuc2/cuc2 double mutant mature embryo showing no GUS activity in the SAM. The expression in the cotyledon tips is maintained. (C) stm-5/stm-5 mature embryo showing a reduction of GUS activity in the SAM. This activity is reduced to two faint spots. The expression in the cotyledon tips is maintained. (D) Wild-type 8-d-old cleared seedling showing GUS activity in the meristem. (E) cuc1-1/cuc1-1 cuc2/cuc2 seedling showing no GUS activity. (F) stm-5/stm-5 seedling exhibiting no GUS activity. Bars = 50 μm.