Shoot apical meristem function in Arabidopsis requires the combined activities of three BEL1-like homeodomain proteins

In plants, most of the above-ground body is formed post-embryonically by the continuous organogenic potential of the shoot apical meristem (SAM). Proper function of the SAM requires maintenance of a delicate balance between the depletion of stem cell daughters into developing primordia and proliferation of the central stem cell population. Here we show that initiation and maintenance of the Arabidopsis SAM, including that of floral meristems, requires the combinatorial action of three members of the BELL-family of TALE homeodomain proteins, ARABIDOPSIS THALIANA HOMEOBOX 1 (ATH1), PENNYWISE (PNY) and POUND-FOOLISH (PNF). All three proteins interact with the KNOX TALE homeodomain protein STM, and combined lesions in ATH1 , PNY and PNF result in a phenocopy of stm mutations. Therefore, we propose that ath1 pny pnf meristem defects result from loss of combinatorial BELL-STM control. Further, we demonstrate that heterodimerization-controlled cellular localization of BELL and KNOX proteins involves a CRM1/exportin-1-mediated nuclear exclusion mechanism that is probably generic to control the activity of BELL and KNOX combinations. We conclude that in animals and plants corresponding mechanisms regulate the activity of TALE homeodomain proteins through controlled nuclear-cytosolic distribution of these proteins.     

Coordination of leaf development via regulation of KNOX1 genes

Class I KNOTTED1-LIKE HOMEOBOX (KNOX1) genes are expressed in the shoot apical meristem (SAM) to effect its formation and maintenance. KNOX1 genes are also involved in leaf shape control throughout angiosperm evolution. Leaves can be classified as either simple or compound, and KNOX1 expression patterns in leaf primordia are highly correlated with leaf shape; in most simple-leafed species, KNOX1 genes are expressed only in the SAM but not in leaf primordia, while in compound-leafed species they are expressed both in the SAM and leaf primordia. How can KNOX1 expression be maintained to a high degree in the SAM, but simultaneously be so variable in leaves? This dichotomy suggests that the processes of leaf and SAM development have been compartmentalized during evolution. Here, we introduce our findings regarding the regulation of expression of SHOOT MERISTEMLESS, a KNOX1 gene, together with a brief review of KNOX1 genes from an evolutionary viewpoint. We also present our findings regarding another aspect of KNOX1 regulation via a protein–protein interaction network involved in the natural variation in leaf shape. Both aspects of KNOX1 regulation could be utilized for fine-tuning leaf morphology during evolution without affecting the essential function of KNOX genes in the shoot.     

Positive autoregulation of a KNOX gene is essential for shoot apical meristem maintenance in rice

Self-maintenance of the shoot apical meristem (SAM), from which aerial organs are formed throughout the life cycle, is crucial in plant development. Class I Knotted1-like homeobox (KNOX) genes restrict cell differentiation and play an indispensable role in maintaining the SAM. However, the mechanism that positively regulates their expression is unknown. Here, we show that expression of a rice (Oryza sativa) KNOX gene, Oryza sativa homeobox1 (OSH1), is positively regulated by direct autoregulation. Interestingly, loss-of-function mutants of OSH1 lose the SAM just after germination but can be rescued to grow until reproductive development when they are regenerated from callus. Double mutants of osh1 and d6, a loss-of-function mutant of OSH15, fail to establish the SAM both in embryogenesis and regeneration. Expression analyses in these mutants reveal that KNOX gene expression is positively regulated by the phytohormone cytokinin and by KNOX genes themselves. We demonstrate that OSH1 directly binds to five KNOX loci, including OSH1 and OSH15, through evolutionarily conserved cis-elements and that the positive autoregulation of OSH1 is indispensable for its own expression and SAM maintenance. Thus, the maintenance of the indeterminate state mediated by positive autoregulation of a KNOX gene is an indispensable mechanism of self-maintenance of the SAM.     

Plant meristems: <Emphasis Type="Italic">CLAVATA3/ESR</Emphasis>-related signaling in the shoot apical meristem and the root apical meristem

The plant meristems, shoot apical meristem (SAM) and root apical meristem (RAM), are unique structures made up of a self-renewing population of undifferentiated pluripotent stem cells. The SAM produces all aerial parts of postembryonic organs, and the RAM promotes the continuous growth of roots. Even though the structures of the SAM and RAM differ, the signaling components required for stem cell maintenance seem to be relatively conserved. Both meristems utilize cell-to-cell communication to maintain proper meristematic activities and meristem organization and to coordinate new organ formation. In SAM, an essential regulatory mechanism for meristem organization is a regulatory loop between WUSCHEL (WUS) and CLAVATA (CLV), which functions in a non-cell-autonomous manner. This intercellular signaling network coordinates the development of the organization center, organ boundaries and distant organs. The CLAVATA3/ESR (CLE)-related genes produce signal peptides, which act non-cell-autonomously in the meristem regulation in SAM. In RAM, it has been suggested that a similar mechanism can regulate meristem maintenance, but these functions are largely unknown. Here, we overview the WUS–CLV signaling network for stem cell maintenance in SAM and a related mechanism in RAM maintenance. We also discuss conservation of the regulatory system for stem cells in various plant species. S. Sawa is the recipient of the BSJ Award for Young Scientist, 2007.     

The role of KNOX genes in the evolution of morphological novelty in Streptocarpus

The genus Streptocarpus comprises species with diverse body plans. Caulescent species produce leaves from a conventional shoot apical meristem (SAM), whereas acaulescent species lack a conventional SAM and produce only a single leaf (the unifoliate form) or clusters of leaves from the base of more mature leaves (the rosulate form). These distinct morphologies reflect fundamental differences in the role of the SAM and the process of leaf specification. A subfamily of KNOTTED-like homeobox (KNOX) genes are known to be important in regulating meristem function and leaf development in model species with conventional morphologies. To test the involvement of KNOX genes in Streptocarpus evolution, two parologous KNOX genes (SSTM1 and SSTM2) were isolated from species with different growth forms. Their phylogenetic analysis suggested a gene duplication before the subgeneric split of Streptocarpus and resolved species relationships, supporting multiple evolutionary origins of the rosulate and unifoliate morphologies. In S. saxorum, a caulescent species with a conventional SAM, KNOX proteins were expressed in the SAM and transiently downregulated in incipient leaf primordia. The ability of acaulescent species to initiate leaves from existing leaves was found to correlate with SSTM1 expression and KNOX protein accumulation in leaves and to reflect genetic differences at two loci. Neither locus corresponded to SSTM1, suggesting that cis-acting differences in SSTM1 regulation were not responsible for evolution of the rosulate and unifoliate forms. However, the involvement of KNOX proteins in leaf formation in rosulate species suggests that they have played an indirect role in the development of morphological diversity in Streptocarpus.     

Characterization of the KNOX class homeobox genes Oskn2 and Oskn3 identified in a collection of cDNA libraries covering the early stages of rice embryogenesis

For identification of genes involved in embryogenesis in the model cereal rice, we have constructed a collection of cDNA libraries of well-defined stages of embryo development before, during and after organ differentiation. Here, we focus on the possible role of KNOX (maize Knotted1-like) class homeobox genes in regulation of rice embryogenesis. Three types of KNOX clones were identified in libraries of early zygotic embryos. Two of these, Oskn2 and Oskn3, encode newly described KNOX genes, whereas the third (Oskn1) corresponds to the previously described OSH1 gene. In situ hybridizations showed that during the early stages of embryo development, all three KNOX genes are expressed in the region where the shoot apical meristem (SAM) is organizing, suggesting that these genes are involved in regulating SAM formation. Whereas OSH1 was previously proposed to function also in SAM maintenance, Oskn3 may be involved in patterning organ positions, as its expression was found to mark the boundaries of different embryonic organs following SAM formation. The expression pattern of Oskn2 suggested an additional role in scutellum and epiblast development. Transgenic expression of Oskn2 and Oskn3 in tobacco further supported their involvement in cell fate determination, like previously reported for Knotted1 and OSH1 ectopic expression. Whereas Oskn3 transformants showed the most pronounced phenotypic effects during vegetative development, Oskn2 transformants showed relatively mild alterations in the vegetative phase but a more severly affected flower morphology. The observation that the KNOX genes produce similar though distinct phenotypic reponses in tobacco, indicates that their gene products act on overlapping but different sets of target genes, or that cell-type specific factors determine their precise action.     

One-leaf plants in the Gesneriaceae: Natural mutants of the typical shoot system

The aerial part of seed plants is called the shoot, which is composed of stems, leaves, and axial buds. These are produced by indeterminate activity in the shoot apical meristem (SAM), whereas the morphogenesis of leaves depends on determinate activity of leaf meristems. However, one-leaf plants in the Gesneriaceae family (eudicots) do not have a typical SAM and do not produce new organs when in the vegetative phase. Instead, they have one cotyledon whose growth is indeterminate. This peculiar development is supported by the groove meristem, which corresponds to the canonical SAM, and the basal meristem, which corresponds to the typical leaf meristem. However, the former does not produce any organ and the latter is active indeterminately. Gene expression and physiological analyses have been conducted in an effort to determine the molecular nature of this peculiar organogenesis. This review summarizes the current understanding of the development of one-leaf plants to provide future perspectives in this field of research.     

The Arabidopsis BEL1-LIKE HOMEODOMAIN proteins SAW1 and SAW2 act redundantly to regulate KNOX expression spatially in leaf margins

In Arabidopsis thaliana, the BEL1-like TALE homeodomain protein family consists of 13 members that form heterodimeric complexes with the Class 1 KNOX TALE homeodomain proteins, including SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP). The BEL1-like protein BELLRINGER (BLR) functions together with STM and BP in the shoot apex to regulate meristem identity and function and to promote correct shoot architecture. We have characterized two additional BEL1-LIKE HOMEODOMAIN (BLH) proteins, SAWTOOTH1 (BLH2/SAW1) and SAWTOOTH2 (BLH4/SAW2) that, in contrast with BLR, are expressed in lateral organs and negatively regulate BP expression. saw1 and saw2 single mutants have no obvious phenotype, but the saw1 saw2 double mutant has increased leaf serrations and revolute margins, indicating that SAW1 and SAW2 act redundantly to limit leaf margin growth. Consistent with this hypothesis, overexpression of SAW1 suppresses overall growth of the plant shoot. BP is ectopically expressed in the leaf serrations of saw1 saw2 double mutants. Ectopic expression of Class 1 KNOX genes in leaves has been observed previously in loss-of-function mutants of ASYMMETRIC LEAVES (AS1). Overexpression of SAW1 in an as1 mutant suppresses the as1 leaf phenotype and reduces ectopic BP leaf expression. Taken together, our data suggest that BLH2/SAW1 and BLH4/SAW2 establish leaf shape by repressing growth in specific subdomains of the leaf at least in part by repressing expression of one or more of the KNOX genes.