Research ArticleResearch Article

KNAT6: An Arabidopsis Homeobox Gene Involved in Meristem Activity and Organ Separation

Enric Belles-Boix, Olivier Hamant, Sarah Melissa Witiak, Halima Morin, Jan Traas and Véronique Pautot
The Plant Cell August 2006, 18 (8) 1900-1907; DOI: https://doi.org/10.1105/tpc.106.041988

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.

INTRODUCTION

In plants, the KNOTTED-like homeobox (KNOX) genes play crucial roles in both embryonic and postembryonic development (Takada and Tasaka, 2002; Hake et al., 2004). In particular, they are required for the initiation and maintenance of the shoot apical meristem (SAM), a group of dividing cells that controls organ production throughout the plant life cycle. Class I of the KNOTTED-like from Arabidopsis thaliana (KNAT) family consists of SHOOT MERISTEMLESS (STM), KNAT1 or BREVIPEDICELLUS (BP), KNAT6, and KNAT2 (Hake et al., 2004). Although STM is required for the formation and maintenance of the SAM (Clark et al., 1996; Endrizzi et al., 1996; Long et al., 1996), BP regulates internode development (Douglas et al., 2002; Venglat et al., 2002; Smith and Hake, 2003) and contributes with STM to SAM maintenance (Byrne et al., 2002). In addition to its role in the SAM, STM interacts with the CUP-SHAPED COTYLEDON (CUC) genes to separate the organs (Takada et al., 2001). During embryogenesis, the CUC gene family plays a crucial role in the establishment of the SAM and in organ separation. The CUC1 and CUC2 genes are redundantly required for cotyledon separation and SAM formation via the activation of the STM gene (Aida et al., 1999; Takada et al., 2001). Further studies demonstrate that the action of CUC1 is mediated by STM: the induction of adventitious SAMs on cotyledons in CUC1 overexpressor lines depends on STM activity (Hibara et al., 2003). In turn, STM is required for the correct expression patterns of CUC2 and to a lesser extent of CUC1 during later stages of embryogenesis (Aida et al., 1999; Takada et al., 2001). The KNAT2 and KNAT6 genes are expressed in the SAM and are the most closely related members of the KNAT family (Dockx et al., 1995; Semiarti et al., 2001). Using an inducible KNAT2 overexpressor line, we previously showed a link between KNOX genes and carpel development (Pautot et al., 2001). A knat2 mutant allele has been reported; however, its development was normal (Byrne et al., 2002). Previous studies using RNA interference lines that downregulate KNAT6 suggested a putative role for KNAT6 in lateral root initiation but did not reveal an obvious role in the shoot (Dean et al., 2004). To investigate the role of KNAT6 and KNAT2 in the SAM, we identified null alleles with T-DNA insertions in each gene and examined their interactions with STM. Here, we show that KNAT6 but not KNAT2 contributes, redundantly with STM, to SAM maintenance and to the establishment of the boundaries via the STM/CUC pathway.

RESULTS

KNAT6 Is Expressed in the SAM Boundaries

To determine the expression pattern of KNAT6, we generated a pKNAT6-GUS (for β-glucuronidase) transcriptional fusion. During embryogenesis, pKNAT6-GUS expression was not detected before the early torpedo stage. In torpedo embryos, expression was restricted to the boundaries between the presumptive SAM and the cotyledons (Figure 1A ). To confirm pKNAT6-GUS expression, we used in situ hybridization. The KNAT6 mRNA was not detected at the globular stage (Figure 1B). It was first detected in the triangular stage embryo, where it appeared in the presumptive SAM (Figure 1C). At the torpedo stage, its expression was detected between the cotyledons and the meristem (Figure 1D). A transverse section of the mature embryo indicated that KNAT6 was expressed between the cotyledons and the meristem and between the cotyledons (Figure 1F). In 8-d-old seedlings, pKNAT6-GUS was expressed in the stipules and marked the boundaries between the SAM and the emerging primordia (see Supplemental Figure 1A online). The expression pattern of KNAT6 is more restricted than that of KNAT2, which is expressed in the rib zone of the meristem (Pautot et al., 2001; see Supplemental Figure 2 online).

Figure 1.
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.

Characterization of knat6 and knat2

To analyze KNAT6 function, two Arabidopsis lines (Columbia [Col-0] ecotype) with T-DNA insertions located in introns 3 and 2 of the KNAT6 gene were characterized. These insertions were named knat6-1 and knat6-2, respectively. A T-DNA insertion line in exon 3 of the KNAT2 gene, named knat2-5, was selected for further analysis. These lines were backcrossed twice to the Col-0 ecotype, and mutant homozygous lines were obtained. To monitor the KNAT6 and KNAT2 transcripts, RNA was isolated from wild-type, knat6-1, knat6-2, and knat2-5 8-d-old seedlings. RT-PCR analysis showed that T-DNA insertions led to null knat6 and knat2 alleles (Figures 2A and 2B ). The development of knat6-1, knat6-2, and knat2-5 homozygous single lines was normal. The analysis of RNA interference lines has suggested that the downregulation of KNAT6 affects lateral root initiation (Dean et al., 2004). Even though we confirmed that KNAT6 is expressed at the site of the lateral root (see Supplemental Figures 1B and 1C online), the development of both null alleles knat6-1 and knat6-2 was normal. To test a potential redundancy with other KNAT genes, we generated the knat6-1 knat2-5 and knat6-2 knat2-5 double mutants. Surprisingly, these double mutants did not show any defect in SAM function (data not shown). This finding indicates that KNAT2 and KNAT6 do not share a redundant function with each other or that other KNAT genes can compensate for the lack of KNAT6 and KNAT2 expression.

Figure 2.
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.

KNAT6, but Not KNAT2, Contributes with STM to SAM Maintenance

The role of BP in the SAM was revealed in the intermediate stm-2 background, showing a redundancy between BP and STM (Byrne et al., 2002). To determine whether that is also the case for KNAT6, we generated the knat6-1 stm-2 double mutant. The stm-2 allele displays a pool of nonorganized meristematic cells and retains the ability to produce vegetative shoots (Clark et al., 1996). Although the stm-2 mutants could produce primordia (Figure 3E ), double knat6-1 stm-2 mutants could not and lacked a SAM (Figure 3F). In addition, knat6-1 stm-2 seedlings exhibited a fusion of cotyledons that extended to the lamina (Figure 3C). In stm mutants, the fusion is restricted to the petiole of the cotyledons, even in the strong alleles (Clark et al., 1996; Long et al., 1996). The knat6-1 stm-2 mutants did not develop further, in contrast with both weak and strong stm mutants, which retain a residual organogenic potential (Clark et al., 1996; Long et al., 1996). The same phenotype was observed with the second knat6 allele (knat6-2 stm-2) or with a strong stm allele (knat6-1 stm-5) (data not shown). Thus, the inactivation of the KNAT6 gene entirely abolished the residual meristematic activity of the stm mutants. By contrast, mutations in KNAT2 showed no such interaction with STM and confirmed previous data (Byrne et al., 2002) (see Supplemental Figure 3 online). We also examined the triple mutant knat6-1 stm-2 knat2-5. The phenotype of these seedlings was identical to that of the double mutant knat6-1 stm-2, showing that KNAT2 does not contribute to the phenotype conferred by knat6-1 stm-2 (see Supplemental Figure 3 online).

Figure 3.
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.

STM and KNAT6 Are Required for the Maintenance of CUC3 Expression

The fusion of the cotyledons observed in the knat6-1 stm-2 double mutant strongly suggests an interaction between KNAT6, STM, and the CUC genes. Previous data showed that the expression patterns of CUC2 and, to a lesser extent, CUC1 were altered in a strong stm mutant. In addition, a mutation in CUC1 or CUC2 enhanced the fusion phenotype of both stm-2 and stm-1 alleles, showing a synergistic interaction between the CUC and STM pathways (Aida et al., 1999). More recently, CUC3 has been shown to have a greater contribution to the separation of the cotyledons than CUC1 and CUC2 (Vroemen et al., 2003). Therefore, we used the WET368 GUS line that mimics the CUC3 gene expression pattern to examine the expression of a boundary marker in the knat6 stm-2 double mutant (Vroemen et al., 2003). In wild-type seedlings, the expression of WET368 GUS is restricted to the boundaries of the cotyledon margins and the boundaries between the SAM and the cotyledons (Vroemen et al., 2003). The knat6-1/knat6-1 stm-2/+ line was crossed to the WET368 GUS homozygous line. F2 plants homozygous for the knat6-1 mutation and for the WET368 GUS marker and heterozygous for the stm-2 mutation were selected (see Methods). GUS assays were performed with siliques from these plants in parallel with the WET368 GUS control line. Embryos that displayed GUS activity corresponded to knat6-1/knat6-1 stm-2/+ and knat6-1/knat6-1 +/+ genotypes, indicating that the expression of WET368 GUS was not altered in the knat6-1 single mutant (Figure 4A ). Embryos that displayed weak GUS activity corresponded to knat6-1/knat6-1 stm-2/stm-2 double mutants (Figure 4B). The F3 progeny were further analyzed. Among 485 seedlings, 351 (72.4%) showed a wild-type phenotype and typical WET368 GUS activity (Figure 4C) and 134 (27.6%) showed the double mutant phenotype. Among the double mutant seedlings, 117 (24.1%) did not show GUS activity (Figure 4E) and 17 (3.5%) displayed weak local residual activity. The WET368 GUS activity was still detected in the stm-2 single mutants (Figure 4D) and in a strong stm-1 allele even in the absence of a proliferating SAM (Vroemen et al., 2003). Thus, the WET368 GUS activity was not maintained in the absence of both STM and KNAT6.

Figure 4.
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.

The Phenotype Conferred by knat6-1 stm-2 Is Slightly Enhanced in a cuc Background

Because we observed residual CUC3 expression in knat6-1 stm-2 embryos, we wondered whether the complete absence of CUC3 expression would aggravate the phenotype conferred by knat6-1 stm-2. We thus constructed and examined the triple mutant knat6-1 stm-2 cuc3-2 (see Methods). The wild-type plants and the majority of the cuc1-1, cuc2, or cuc3-2 homozygous single mutants have separate cotyledons (Aida et al., 1999; Vroemen et al., 2003). By contrast, double mutant combinations of CUC1, CUC2, or CUC3 lead to cup-shaped seedlings (Aida et al., 1999; Vroemen et al., 2003). The majority (90%) of the knat6-1 stm-2 cuc3-2 triple mutants exhibited a stronger cotyledon fusion than the knat6-1 stm-2 double mutant (Figures 5I , 5L, 5M, and 5P). This finding reflects the contribution of the CUC3 residual activity present in the knat6-1 stm-2 embryo. The stronger fusion phenotype in the cuc1-1 cuc2 double mutant than in the knat6 stm-2 background suggests that the inactivation of both KNAT6 and STM is also not sufficient to completely repress CUC1 and CUC2 (Figures 5B, 5F, 5I, and 5M). To determine whether the complete absence of CUC1 or CUC2 expression would aggravate the phenotype conferred by knat6-1 stm-2, we examined the knat6-1 stm-2 cuc1-1 and knat6-1 stm-2 cuc2 triple mutants. As shown in Figures 5J to 5L, the fusion observed in the absence of CUC2 was less severe than that observed in the absence of CUC1 or CUC3. In addition, only 15% of the knat6-1 stm-2 cuc2 and 21% of the knat6-1 stm-2 cuc1-1 seedlings showed a stronger fusion than that of the knat6-1 stm-2 double mutant. This could suggest that the inactivation of both KNAT6 and STM has a stronger impact on the expression of CUC2 and CUC1 than on CUC3. Together, these results suggest that KNAT6 and STM are corequired for the maintenance of CUC gene expression during late embryogenesis.

Figure 5.
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]).

Activation of KNAT6 Is CUC Dependent

We have shown that CUC3 expression was altered in the knat6 stm-2 background. We wondered next whether, conversely, KNAT6 expression was impaired in a cuc background. To this purpose, the homozygous pKNAT6-GUS line was crossed to a line homozygous for the cuc1-1 mutation and heterozygous for the cuc2 mutation. F2 plants homozygous for the pKNAT6-GUS construct, homozygous for the cuc1-1 mutation, and heterozygous for the cuc2 mutation were selected. In the F3 progeny, among 195 seedlings, 149 (76.4%) showed a wild-type phenotype and 46 (23.6%) showed a cup-shaped phenotype. The cup-shaped seedlings corresponded to cuc1-1/cuc1-1 cuc2/cuc2 double mutants. The wild-type plants exhibited GUS activity similar to that of the control, whereas the cup-shaped seedlings did not exhibit GUS activity (Figures 6D and 6E ). F2 plants homozygous for the pKNAT6-GUS construct, heterozygous for the cuc1-1 mutation, and homozygous the cuc2 mutation from the progeny that segregated the cup-shaped phenotype were selected by PCR. In the F3 progeny, among 250 seedlings, 168 (67.2%) showed a wild-type phenotype, 17 (6.8%) showed a heart-shaped phenotype, and 65 (26.0%) showed a cup-shaped phenotype. The low percentage of the heart-shaped seedlings corresponded to the cuc1/+ cuc2/cuc2 genotype (Aida et al., 1997), and the cup-shaped seedlings corresponded to the cuc1-1 cuc2 double mutant genotype. The wild type exhibited GUS activity in the meristem, whereas the cup-shaped seedlings did not show GUS activity (Figures 6D and 6E). During embryogenesis, KNAT6-GUS activity was never detected in cuc1-1 cuc2 double mutant embryos except in the tip of the cotyledons (Figure 6B). Collectively, the analysis of these two F3 progeny indicates that CUC1 and CUC2 are redundantly required to activate the KNAT6 gene during embryogenesis. These data are consistent with the fact that KNAT6 is expressed after the CUC genes during embryogenesis.

Figure 6.
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.

Maintenance of KNAT6 Expression Is STM Dependent

We next examined KNAT6 expression in a strong stm mutant. For this purpose, the pKNAT6-GUS homozygous line was crossed to a line that was heterozygous for the stm-5 strong allele. F2 plants homozygous for the pKNAT6-GUS construct and heterozygous for the stm-5 mutation were selected. GUS assays were performed on siliques from these F2 plants. Although the expression pattern was normal in the wild-type and heterozygous embryos, the homozygous stm-5 embryos showed a reduction of GUS activity in the SAM (Figure 6C). GUS activity was still detected at the tips of the cotyledons. We next examined pKNAT6-GUS expression in 8-d-old seedlings in the F3 progeny. All of the seedlings with a wild-type phenotype exhibited GUS activity similar to that of the control (Figure 6D), whereas the majority of the stm-5 seedlings did not show GUS activity (Figure 6F). The residual GUS activity seen in stm-5 embryos was not further maintained during development. These data suggest that STM maintains the activity of KNAT6 in the SAM.

DISCUSSION

KNAT6, a Gene Involved in Meristem Maintenance and Boundary Establishment

The absence of meristematic defects in the knat6 single mutant confirms the major role of STM in the SAM. However, our data showed that the inactivation of KNAT6 entirely abolishes the residual meristematic activity of the stm-2 weak allele and impairs organ separation, revealing a role for KNAT6 in the SAM. Interestingly, no such phenotype was observed in knat2 stm or in bp (knat1) stm double mutants. The inactivation of KNAT2 did not enhance the phenotype of the weak stm allele or the knat6 stm-2 double mutant. By contrast, the inactivation of BP enhances a weak allele of stm without affecting the fusion phenotype of the mutant (Byrne et al., 2002). The specific contribution of KNAT6 in organ separation is consistent with its expression pattern because it matches the expression domain of the CUC genes in late embryogenesis: in mature embryos, the expression of KNAT6 was detected between the cotyledons and the meristem and between the cotyledons where the fusion takes place in the knat6 stm double mutant. Thus, to date, KNAT6 is with STM the only class I KNAT gene involved in boundary establishment during embryogenesis.

Position of KNAT6 in the STM/CUC Network

The KNAT6 mRNA was first detected at the triangular stage. Thus, KNAT6 is expressed later than STM and CUC genes. In contrast with other meristematic genes such as BP or CLAVATA3 that are still expressed in the absence of a proliferating SAM (Brand et al., 2002; Hay et al., 2002), the maintenance of KNAT6 expression required STM. Therefore, KNAT6 is likely a downstream effector in the STM pathway in the embryo. However, the enhanced fusion of the cotyledons and the lack of a SAM in the knat6 stm double mutant indicate that the residual activity of KNAT6 in the stm embryo prevents the fusion of the cotyledon lamina. This phenotype led us to examine the link between KNAT6, STM, and the CUC genes. Our analysis indicates that the expression of CUC3 was not altered in the knat6-1 single mutant. By contrast, CUC3 expression was reduced in knat6-1 stm-2 embryos and absent in the double mutant seedlings. Examination of the knat6-1 stm-2 cuc3-2 triple mutant supported these data: the loss of function of CUC3 enhanced the fusion of the knat6-1 stm-2 phenotype. Additionally, the fusions observed in the absence of CUC1 or CUC2 were less severe than that observed in the absence of CUC3. This finding confirmed the greater contribution of CUC3 to the separation of cotyledons (Vroemen et al., 2003). We never observed in any of the triple mutants the typical cup-shaped phenotype, suggesting that the expression of the two remaining CUC genes was not abolished completely. This is in agreement with the fact that, in the wild type, KNAT6 is expressed after STM and the CUC genes. Together, our results show that KNAT6 function is integrated in a network comprising STM and the CUC genes to regulate organ separation and to maintain the SAM. KNAT2, the closest member to KNAT6, did not display such a role.

The Function of KNAT2 Is Still Unknown

It is possible that the function of KNAT2 could be revealed in a different background. In previous work, we showed that in the presence of ethylene, KNAT2 expression is restricted to the boundaries between the SAM and the cotyledons (Hamant et al., 2002). Therefore, it is possible that a redundancy between KNAT2 and KNAT6 could be revealed by modulating some effectors of the ethylene transduction pathway. Furthermore, the BP and STM proteins interact with the BELLRINGER (BLR) protein to control inflorescence stem growth (Byrne et al., 2003; Smith and Hake, 2003; Bhatt et al., 2004). Although the loss of function of KNAT2 alone has no phenotypic effect on the phenotype of the blr mutant (Byrne et al., 2003), it is possible that KNAT2 interacts with other KNAT and BELL family members. More recently, a study reported another plant protein family, the OVATE family, that interacts with the TALE KNOX and BELL proteins to control their intracellular localization (Hackbusch et al., 2005). This study indicates potential functional redundancy between the members of these families. Further genetic studies are required to determine whether KNAT2 contributes to the SAM.

METHODS

Plant Material and Growth Conditions

Arabidopsis thaliana plants were grown either on soil or in vitro as described by Hamant et al. (2002). The Arabidopsis ecotypes Landsberg erecta (Ler), Col-0, and Wassilewkija (Ws) were used in this study. Mutant alleles of knat6-1, knat6-2, and knat2-5 were obtained from the Nottingham Arabidopsis Stock Centre (http://nasc.nott.ac.uk). Mutant alleles of stm-2 (weak allele, Ler ecotype) and stm-5 (strong allele, Ler ecotype) were provided by Thomas Laux. The cuc1-1 (Ler) and cuc2 (Ler) mutant alleles were provided by Mitsuhiro Aida (Aida et al., 1997; Takada et al., 2001). The cuc3-2 (Ws) mutant allele and the WET368 GUS line (Ler) that expresses the CUC3 gene were provided by Casper W. Vroemen (Vroemen et al., 2003). The translational KNAT2-GUS fusion has been described (Dockx et al., 1995).

Insertion Mutant Screen and RT-PCR Analysis

Two T-DNA insertion lines in KNAT6 (At1g23380) were identified using the signal T-DNA express Arabidopsis gene-mapping tool (http://signal.salk.edu/cgibin/T-DNAexpress) (Alonso et al., 2003). To check the T-DNA insertion positions, genomic DNA from mutant plants was amplified with the left border Lba1 T-DNA primer (5′-TGGTTCACGTAGTGGGCCATCG-3′) and the KNAT6 specific primers KNAT6-03 (5′-GAAGATAAACCCTAGCTACAAG-3′) for N547931 and KNAT6-40 (5′-ACAATTTCCATTCGGCCGGTGATT-3′) for N554482. PCR products were sequenced using dye terminator cycle sequencing (Applied Biosystems). The insertion line N547931, with a T-DNA insertion at position +1740 from the ATG (intron 3), was named knat6-1. The insertion line N554482, with a T-DNA insertion at position +647 from the ATG (intron 2), was named knat6-2. Nottingham Arabidopsis Stock Centre line N59837, which has a T-DNA insertion in KNAT2 (At1g70510), was named knat2-5. PCR amplification of the flanking sequences using the Lba1 T-DNA primer and either the forward KNAT2 specific primer 4K1 (5′-CGCTTCTCATCCTTTGTATC-3′) or the reverse KNAT2 specific primer k11 (5′-GATTCAATACGATACGATCC-3′) showed that the insertion contains two T-DNAs in reverse tandem at position +758 from the ATG (exon 3).

Total RNA was extracted using Tri reagent (Sigma-Aldrich), treated with DNase I (Invitrogen), and purified through RNeasy mini columns (Qiagen). cDNA was synthesized using 2 μg of total RNA and 50 units of SuperScript II RNase H− reverse transcriptase (Invitrogen) according to the manufacturer's instructions. PCR was performed using the KNAT6-15 (5′-AGATAAGTCGGTTCTGATGATG-3′) and KNAT6-18 (5′-GATCCACTAGAATCATCCATCATAGCGAA-3′) specific primers. After 38 cycles, PCR products were subjected to DNA gel blot hybridization using a 33P-labeled KNAT6 specific probe. To monitor the KNAT2 mRNA levels, RT-PCR was performed using the KNAT2 specific primers 4K1 (5′-CGCTTCTCATCCTTTGTATC-3′) and 6K1 (5′-CCGCTGCTATGTCATCATC-3′). After 40 PCR cycles, PCR products were subjected to DNA gel blot hybridization using a 33P-labeled KNAT2 specific probe. Control amplification was performed using the forward (5′-TCCCAGAATCGCTAAGATTGCC-3′) and reverse (5′-CCTTTCCCTTAAGCTCTG-3′) specific primers of APT1 (At3g18780) and 38 cycles.

Construction of the pKNAT6-GUS Fusion

A 3.8-kb genomic fragment containing the KNAT6 promoter was amplified from the Col-0 ecotype using the high-fidelity Pfu polymerase (Stratagene) and KNAT6-38F (5′-CTAGTCTAGACTAGTATAAGGCTTGATTATGAACAACAAAGAC-3′) and KNAT6-39R (5′-CTAGTCTAGACTAGTCCATCCATAAGTATAAGATCTCCGGTAA-3′) primers. Each primer contained a XbaI restriction site allowing the PCR product to be cloned. The promoter was sequenced and subcloned upstream of the GUS coding sequence in the binary vector pBI101-1 (Clontech). The binary vector harboring the pKNAT6-GUS transcriptional fusion was introduced into Agrobacterium tumefaciens C58. Arabidopsis (Ws ecotype) was transformed by floral dip (Clough and Bent, 1998). Transformants were selected on medium containing 50 mg/L kanamycin. Homozygous lines for the pKNAT6-GUS construct were selected.

In Situ Hybridization

In situ hybridizations were performed as described previously (Vernoux et al., 2000). The KNAT6 antisense probe was generated using the pDONOR201 vector that contains the KNAT6 full-length cDNA as a template and using KNAT6-34b (5′-GGGAGCTCATGTACAATTTCCATTC-3′) and KNAT6-04T7 (5′-TGTAATACGACTCACTATAGGGCTCATTCCTCGGTAAAGAA-3′), which incorporates a binding site for T7 polymerase, as primers. A PCR product that did not contain the homeobox gave the same expression pattern. Hybridization was performed at 43°C. In situ hybridization of KNAT2 mRNA was described previously (Pautot et al., 2001).

Plant Genetics

To construct the knat6-1 stm-2 double mutants, plants homozygous for knat6-1 were crossed to plants heterozygous for stm-2. A novel phenotype segregated in the F2 progeny in the expected 1:16 ratio. The same phenotype was observed when using other allelic combinations: the second allele of knat6 (knat6-2 stm-2) or a null allele of stm (knat6-1 stm-5). F2 plants homozygous for knat6-1 and heterozygous for stm-2 were selected by PCR analysis. To follow the stm-2 mutation, we generated CAPS primers. The stm-2 and wild-type Ler alleles were sequenced. The sequence of the stm-2 allele revealed a point mutation (G to A) at position +2053 from ATG (+906 from ATG in the cDNA). This conversion generates a new BccI restriction site and introduces a premature stop codon. This truncated protein has 252 amino acids instead of the 383 amino acids of the wild-type protein. Thus, the stm-2 genotype can be followed by PCR amplification and enzymatic restriction. Genomic DNA was amplified with the STM specific primers STM1 (5′-GAGACAGCAATTGATAGGAACAAT-3′) and STMR1 (5′-ATGGTGGAGGAGATGTGATCC-3′). The PCR program used was as follows: denaturation at 95°C for 3 min; 38 cycles of priming at 93°C for 20 s and annealing at 58°C for 20 s; and extension at 72°C for 40 s. The amplification produces a fragment of 1077 bp (Col-0) or 1110 bp (Ler and stm-2). The PCR mixture was desalted during 20 min with Millipore dialysis membranes and then digested at 37°C for 3 h with 5 units of the restriction endonuclease BccI. Digestion of the wild-type Col-0 STM allele generates two fragments of 327 and 752 bp. Digestion of the wild-type Ler STM allele generates two fragments of 330 and 780 bp. Digestion of the mutant stm-2 allele generates three fragments of 211, 330, and 569 bp. The double mutant was confirmed by PCR. In the F3 progeny from a selfed knat6-1/knat6-1 stm-2/+ plant, a total of 303 seedlings were scored: 221 (73%) were wild type and 82 (27%) were double mutants. This ratio corresponds to the expected 3:1 ratio.

To generate the knat6-1 stm-2 cuc1-1 triple mutants, plants homozygous for knat6-1 and heterozygous for stm-2 were crossed to plants homozygous for cuc1-1. F2 plants homozygous for knat6-1 and cuc1 and heterozygous for stm-2 were selected by PCR analysis (as described above and in Takada et al., 2001). In the F3 progeny from a selfed knat6-1/knat6-1 cuc1-1/cuc1-1 stm-2/+ plant, 239 seedlings were scored: 77.2% of seedlings showed the wild-type phenotype and 22.8% showed a mutant phenotype. Among the mutant seedlings, 79% showed a typical double mutant phenotype and 21% exhibited an enhanced cotyledon fusion.

To generate the knat6-1 stm-2 cuc2 triple mutants, plants homozygous for knat6-1 and heterozygous for stm-2 were crossed to plants homozygous for cuc2. F2 plants homozygous for knat6-1 and cuc2 and heterozygous for stm-2 were selected by PCR analysis (as described above and in Takada et al., 2001). In the F3 progeny from a selfed knat6-1/knat6-1 cuc2/cuc2 stm-2/+ plant, 172 seedlings were scored: 76.7% of seedlings showed the wild-type phenotype and 23.3% showed a mutant phenotype. Among the mutant seedlings, 85% showed a typical double mutant phenotype and 15% exhibited a slight enhanced cotyledon fusion.

To generate the knat6-1 stm-2 cuc3-2 triple mutants, plants homozygous for knat6-1 and heterozygous for stm-2 were crossed to plants homozygous for cuc3-2. F2 plants homozygous for knat6-1 and cuc3-2 and heterozygous for stm-2 were selected by PCR analysis. To detect the cuc3-2 allele, the TAG5 T-DNA primer (5′-CTACAAATTGCCTTTTCTTATCGAC-3′) and the CUC3 specific primer (5′-ATGATGCTTGCGGTGGAAGA-3′) were used. In the F3 progeny from a selfed knat6-1/knat6-1 cuc3-2/cuc3-2 stm-2/+ plant, 254 seedlings were scored: 73.6% of seedlings showed the wild-type phenotype and 26.4% showed a mutant phenotype. Among the mutant seedlings, 10% showed a typical double mutant phenotype and 90% exhibited an enhanced cotyledon fusion.

To examine the expression of CUC3 in the knat6-1 and knat6-1 stm-2 mutants, the homozygous WET368 GUS line was crossed to the line homozygous for the knat6-1 mutation and heterozygous for the stm-2 allele. The WET368 GUS line is a hypomorph allele of CUC3, because the CUC3 mRNA level is reduced to 20% of the wild-type level (Vroemen et al., 2003). When we examined the F2 progeny, the knat6-1 stm-2 double mutant seedlings never exhibited GUS activity. This finding shows that WET368 GUS is downregulated in the knat6-1 stm-2 double mutant even in the heterozygous seedlings for the WET368 GUS marker. By contrast, GUS activity was still detected in the stm-2 single mutants. F2 plants were analyzed by PCR to select plants homozygous for knat6-1 and heterozygous for stm-2. The F3 progeny of these F2 plants were examined for GUS activity to select plants homozygous for the WET368 GUS marker.

To examine the expression of KNAT6 in cuc1 and cuc2 mutants, the homozygous pKNAT6-GUS line was crossed to a line homozygous for the cuc1-1 mutation and heterozygous for the cuc2 mutation. Two types of F1 progeny were obtained: those that segregated the cup-shaped phenotype in F2 and those that did not. F2 plants were analyzed by PCR to genotype the cuc1-1 and cuc2 mutations. An F2 plant homozygous for the cuc1 mutation and heterozygous for the cuc2 mutation and one heterozygous for the cuc1 mutation and homozygous for the cuc2 mutation were selected. The F3 progeny of these F2 plants were examined for GUS activity to select plants homozygous for the pKNAT6-GUS construct.

To examine the expression of KNAT6 in a strong stm mutant, the pKNAT6-GUS homozygous line was crossed to a line that was heterozygous for the stm-5 allele. F2 plants homozygous for the pKNAT6-GUS construct and heterozygous for the stm-5 mutation were selected based on analysis of the F3 progeny. Among 342 F3 seedlings, 261 (76.3%) displayed the wild-type phenotype and exhibited GUS activity and 81 (23.7%) displayed an stm phenotype. Of these 81 stm seedlings, 78 showed no GUS activity and 3 exhibited very weak KNAT6-GUS activity in the apical part.

Histological Analysis

Tissue clearing, GUS staining, and Nomarski microscopy were performed as described previously (Pautot et al., 2001).

Accession Numbers

The Arabidopsis Genome Initiative numbers for KNAT2 and KNAT6 are At1g70510 and At1g23380, respectively.

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. pKNAT6-GUS Expression.

  • Supplemental Figure 2. KNAT2 Expression in Embryo.

  • Supplemental Figure 3. Phenotypes of the knat2-5 stm-2 and knat2-5 knat6-1 stm-2 Mutants.

Acknowledgments

We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. We thank Bruno Letarnec for greenhouse management and Patrick Laufs and Laura Ragni for critical reading of the manuscript. E.B.-B. was funded by the Region Ile de France and the Institut National de la Recherche Agronomique. Part of this work was supported by the Regulatory Gene Initiative in Arabidopsis program. S.M.W. was supported by the Sustainable Crop Protection in Agriculture Program at Pennsylvania State University.

Footnotes

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Véronique Pautot (pautot{at}versailles.inra.fr).

  • Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.106.041988.

  • 1 These authors contributed equally to this work.

  • 2 Current address: Laboratoire Reproduction et Développement des Plantes, Ecole Normale Supérieure Lyon, 46 Allée d'Italie, 69364 Lyon Cedex 07, France.

  • 3 Current address: 122 Chemical Ecology Lab, Orchard Road, University Park, PA 16802.

  • [W] Online version contains Web-only data.

  • Received February 17, 2006.
  • Revised April 23, 2006.
  • Accepted May 19, 2006.
  • Published June 23, 2006.

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