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

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.     

The NAC domain mediates functional specificity of CUP-SHAPED COTYLEDON proteins

In higher plants, although several genes involved in shoot apical meristem (SAM) formation and organ separation have been isolated, the molecular mechanisms by which they function are largely unknown. CUP-SHAPED COTYLEDON (CUC) 1 and CUC2 are examples of two such genes that encode the NAC domain proteins. This study investigated the molecular basis for their activities. Nuclear localization assays indicated that green fluorescent protein (GFP)-CUC proteins accumulate in the nucleus. Yeast one-hybrid and transient expression assays demonstrated that the C-terminal domain (CTD) of the CUC has transactivation activity. Domain-swapping experiments revealed that the functional specificity of the CUC for promoting adventitious shoot formation resides in the highly conserved NAC domain, not in the CTD in which motifs specific to the CUC subfamily are located. Taken together, these observations suggest that CUC proteins transactivate the target genes involved in SAM formation and organ separation through a specific interaction between the NAC domain and the promoter region of the target genes.     

Expression of PaNAC01, a Picea abies CUP-SHAPED COTYLEDON orthologue, is regulated by polar auxin transport and associated with differentiation of the shoot apical meristem and formation of separated cotyledons

Background and Aims During embryo development in most gymnosperms, the establishment of the shoot apical meristem (SAM) occurs concomitantly with the formation of a crown of cotyledons surrounding the SAM. It has previously been shown that the differentiation of cotyledons in somatic embryos of Picea abies is dependent on polar auxin transport (PAT). In the angiosperm model plant, Arabidopsis thaliana, the establishment of cotyledonary boundaries and the embryonal SAM is dependent on PAT and the expression of the CUP-SHAPED COTYLEDON (CUC) genes, which belong to the large NAC gene family. The aim of this study was to characterize CUC-like genes in a gymnosperm, and to elucidate their expression during SAM and cotyledon differentiation, and in response to PAT. Methods Sixteen Picea glauca NAC sequences were identified in GenBank and deployed to different clades within the NAC gene family using maximum parsimony analysis and Bayesian inference. Motifs conserved between angiosperms and gymnosperms were analysed using the motif discovery tool MEME. Expression profiles during embryo development were produced using quantitative real-time PCR. Protein conservation was analysed by introducing a P. abies CUC orthologue into the A. thaliana cuc1cuc2 double mutant. Key Results Two full-length CUC-like cDNAs denoted PaNAC01 and PaNAC02 were cloned from P. abies. PaNAC01, but not PaNAC02, harbours previously characterized functional motifs in CUC1 and CUC2. The expression profile of PaNAC01 showed that the gene is PAT regulated and associated with SAM differentiation and cotyledon formation. Furthermore, PaNAC01 could functionally substitute for CUC2 in the A. thaliana cuc1cuc2 double mutant. Conclusions The results show that CUC-like genes with distinct signature motifs existed before the separation of angiosperms and gymnosperms approx. 300 million years ago, and suggest a conserved function between PaNAC01 and CUC1/CUC2.     

SHOOT MERISTEMLESS trafficking controls axillary meristem formation,meristem size and organ boundaries in Arabidopsis

The shoot stem cell niche, contained within the shoot apical meristem (SAM) is maintained in Arabidopsis by the homeodomain protein SHOOT MERISTEMLESS (STM). STM is a mobile protein that traffics cell‐to‐cell, presumably through plasmodesmata. In maize, the STM homolog KNOTTED1 shows clear differences between mRNA and protein localization domains in the SAM. However, the STM mRNA and protein localization domains are not obviously different in Arabidopsis, and the functional relevance of STM mobility is unknown. Using a non‐mobile version of STM (2xNLS‐YFP‐STM), we show that STM mobility is required to suppress axillary meristem formation during embryogenesis, to maintain meristem size, and to precisely specify organ boundaries throughout development. STM and organ boundary genes CUP SHAPED COTYLEDON1 (CUC1), CUC2 and CUC3 regulate each other during embryogenesis to establish the embryonic SAM and to specify cotyledon boundaries, and STM controls CUC expression post‐embryonically at organ boundary domains. We show that organ boundary specification by correct spatial expression of CUC genes requires STM mobility in the meristem. Our data suggest that STM mobility is critical for its normal function in shoot stem cell control.     

CUC1 gene activates the expression of SAM-related genes to induce adventitious shoot formation

CUP-SHAPED COTYLEDON (CUC)1 encodes members of the NAC family. These are functionally redundant genes that are involved in shoot apical meristem (SAM) formation and cotyledon separation during embryogenesis in Arabidopsis. We analyzed transgenic plants overexpressing CUC1 (35S::CUC1). The cotyledons of these transgenic seedlings regularly had two basal lobes, small and round epidermal cells between the sinuses, and adventitious SAMs on the adaxial surface of this region. This suggests that CUC1 promotes adventitious SAM formation by maintaining epidermal cells in an undifferentiated state. In 35S::CUC1 cotyledons, the class I knotted-like homeobox (KNOX) genes, including SHOOT MERISTEMLESS (STM) and BREVIPEDICELLUS (BP), which are involved in SAM formation and/or maintenance, were ectopically expressed before adventitious SAM formation. In stm mutants, ectopic expression of CUC1 could not induce adventitious SAMs, whereas they continued to be observed in bp mutants. These results suggest that STM, but not BP, is necessary for the formation of adventitious SAMs in 35S::CUC1 cotyledons. Furthermore, we examined the relationship between CUC1 and ASYMMETRIC LEAVES (AS)1 and AS2. The as1 and as2 mutations genetically enhance 35S::CUC1 phenotypes even in the absence of STM function. Interestingly, the as1 mutation can partially rescue the mutant vegetative development phenotypes in the cuc1 cuc2 double mutant. Our results suggest that CUC1 positively regulates SAM formation not only through STM but also through an STM-independent pathway that is negatively regulated by AS1 and AS2.     

Developmental events and shoot apical meristem gene expression patterns during shoot development in Arabidopsis thaliana

Critical developmental and gene expression profiles were charted during the formation of shoots from root explants in Arabidopsis tissue culture. Shoot organogenesis is a two-step process involving pre-incubation on an auxin-rich callus induction medium (CIM) during which time root explants acquire competence to form shoots during subsequent incubation on a cytokinin-rich shoot induction medium (SIM). At a histological level, the organization of shoot apical meristems (SAMs) appears to occur during incubation on SIM about the time of shoot commitment, i.e. the transition from hormone-dependent to hormone-independent shoot development. Genes involved in SAM formation, such as SHOOTMERISTEMLESS (STM) and CLAVATA1 (CLV1), were upregulated at about the time of shoot commitment, while WUSCHEL (WUS) was upregulated somewhat earlier. Genes required for STM expression, such as CUP-SHAPED COTYLEDON 1 and 2 (CUC1 and 2) were upregulated prior to shoot commitment. Gene expression patterns were determined for two GFP enhancer trap lines with tissue-specific expression in the SAM, including one line reporting on CUC1 expression. CUC1 was generally expressed in callus tissue during early incubation on SIM, but later CUC1 was expressed more locally in presumptive sites of shoot formation. In contrast, the expression pattern of the enhancer trap lines during zygotic embryogenesis was more localized to the presumptive SAM even in early stages of embryogenesis.     

Identification of novel meristem factors involved in shoot regeneration through the analysis of temperature-sensitive mutants of Arabidopsis

Adventitious organogenesis in plant tissue culture involves de novo formation of apical meristems and should therefore provide important information about the fundamentals of meristem gene networks. We identified novel factors required for neoformation of the shoot apical meristem (SAM) through an analysis of shoot regeneration in root initiation defective3 ( rid3 ) and root growth defective3 ( rgd3 ) temperature-sensitive mutants of Arabidopsis. After induction of callus to regenerate shoots, cell division soon ceased and was then reactivated locally in the surface region, resulting in formation of mounds of dense cells in which adventitious-bud SAMs were eventually constructed. The rgd3 mutation inhibited reactivation of cell division and suppressed expression of CUP-SHAPED COTYLEDON1 ( CUC1 ), CUC2 and SHOOT MERISTEMLESS ( STM ). In contrast, the rid3 mutation caused excess ill-controlled cell division on the callus surface. This was intimately related to enhanced and broadened expression of CUC1 . Positional cloning revealed that the RGD3 and RID3 genes encode BTAF1 (a kind of TATA-binding protein-associated factor) and an uncharacterized WD-40 repeat protein, respectively. In the early stages of shoot regeneration, RGD3 was expressed (as was CUC1 ) in the developing cell mounds, whereas RID3 was expressed outside the cell mounds. When RID3 was over-expressed artificially, the expression levels of CUC1 and STM were significantly reduced. Taken together, these findings show that both negative regulation by RID3 and positive regulation by RGD3 of the CUC–STM pathway participate in proper control of cell division as a prerequisite for SAM neoformation.     

The GURKE gene encoding an acetyl-CoA carboxylase is required for partitioning the embryo apex into three subregions in Arabidopsis

In Arabidopsis, three major regions, which ultimately develop into the two cotyledons, the cotyledon boundaries and the shoot apical meristem (SAM), are formed at the apex of the globular stage embryo. To reveal the molecular mechanism underlying this pattern formation, we isolated a cotyledon-defective mutant from EMS mutagenized lines. This mutant completely lacks cotyledons in the most severe cases, and is allelic to gurke (gk), which was previously reported as a mutant defective in apical patterning of the embryo. To evaluate the morphological effects of the mutation in the GK gene, we investigated the expression patterns in gk embryos of SHOOT MERISTEMLESS (STM), AINTEGUMENTA (ANT) and CUP-SHAPED COTYLEDON1 (CUC1), which are markers of the SAM, cotyledons and cotyledon boundaries, respectively. Expression of all these genes largely overlapped in gk, suggesting a failure to partition the apex of the embryo into the three subregions. Enlargement of the CUC1 expression domain was also observed and may explain the inhibition of cotyledon development in gk. Moreover, we cloned the GK gene, and confirmed that it encodes ACC1, an acetyl-CoA carboxylase which catalyzes malonyl-CoA synthesis. Our results suggest that metabolites derived from malonyl-CoA are required for partitioning of the apical part of the embryo.     

Involvement of CUP-SHAPED COTYLEDON genes in gynoecium and ovule development in Arabidopsis thaliana

When mutations in CUP-SHAPED COTYLEDON1 (CUC1) and CUC2 are combined, severe defects involving fusion of sepals and of stamens occur in Arabidopsis flowers. In addition, septa of gynoecia do not fuse along the length of the ovaries and many ovules have their growth arrested. CUC2 is expressed at the tips of septal primordia during gynoecium development and at the boundary between nucellus and chalaza during ovule development. These expression patterns are partially consistent with the phenotype of the mutant gynoecium. CUC2 mRNA is also shown to be expressed at the boundaries between meristems and organ primordia during both the vegetative and reproductive phases. This expression pattern indicates that CUC2 is generally involved in organ separation in shoot and floral meristems.