The bHLH protein ROX acts in concert with RAX1 and LAS to modulate axillary meristem formation in Arabidopsis

During post-embryonic shoot development, new meristems are initiated in the axils of leaves. They produce secondary axes of growth that determine morphological plasticity and reproductive efficiency in higher plants. In this study, we describe the role of the bHLH-protein-encoding Arabidopsis gene REGULATOR OF AXILLARY MERISTEM FORMATION (ROX), which is the ortholog of the branching regulators LAX PANICLE1 (LAX1) in rice and barren stalk1 (ba1) in maize. rox mutants display compromised axillary bud formation during vegetative shoot development, and combination of rox mutants with mutations in RAX1 and LAS, two key regulators of axillary meristem initiation, enhances their branching defects. In contrast to lax1 and ba1, flower development is unaffected in rox mutants. Over-expression of ROX leads to formation of accessory side shoots. ROX mRNA accumulates at the adaxial boundary of leaf and flower primordia. However, in the vegetative phase, axillary meristems initiate after ROX expression has terminated, suggesting an indirect role for ROX in meristem formation. During vegetative development, ROX expression is dependent on RAX1 and LAS activity, and all three genes act in concert to modulate axillary meristem formation.     

Phenotypical and structural characterization of the Arabidopsis mutant involved in shoot apical meristem

An Arabidopsis mutant induced by T-DNA insertion was studied with respect to its phenotype, micro-structure of shoot apical meristem (SAM) and histo-chemical localization of the GUS gene in comparison with the wild type. Phenotypical observation found that the mutant exhibited a dwarf phenotype with smaller organs (such as smaller leaves, shorter petioles), and slower development and flowering time compared to the wild type. Optical microscopic analysis of the mutant showed that it had a smaller and more flattened SAM, with reduced cell layers and a shortened distance between two leaf primordia compared with the wild type. In addi-tion, analysis of the histo-chemical localization of the GUS gene revealed that it was specifically expressed in the SAM and the vascular tissue of the mutant, which suggests that the gene trapped by T-DNA may function in the SAM, and T-DNA insertion could influence the functional activity of the related gene in the mutant, lead-ing to alterations in the SAM and a series of phenotypes in the mutant.     

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.     

Enhanced cytokinin degradation in leaf primordia of transgenic Arabidopsis plants reduces leaf size and shoot organ primordia formation

The plant hormone cytokinin is a key morphogenic factor controlling cell division and differentiation, and thus the formation and growth rate of organs during a plant's life cycle. In order to explore the relevance of cytokinin during the initial phase of leaf primordia formation and its impact on subsequent leaf development, we increased cytokinin degradation in young shoot organ primordia of Arabidopsis thaliana by expressing a cytokinin oxidase/dehydrogenase (CKX) gene under control of the AINTEGUMENTA (ANT) promoter. The final leaf size in ANT:CKX3 plants was reduced to ∼27% of the wild-type size and the number of epidermal cells was reduced to ∼12% of the wild type. Kinematic analysis revealed that cell proliferation ceased earlier and cell expansion was accelerated in ANT:CKX3 leaves, demonstrating that cytokinin controls the duration of the proliferation phase by delaying the onset of cell differentiation. The reduction of the cell number was partially compensated by an increased cell expansion. Interestingly, ANT:CKX3 leaf cells became about 60% larger than those of 35S:CKX3 leaves, indicating that cytokinin has an important function during cell expansion as well. Furthermore, ANT:CKX3 expression significantly reduced the capacity of both the vegetative as well as the generative shoot apical meristem to initiate the formation of new leaves and flowers, respectively. We therefore hypothesize that the cytokinin content in organ primordia is important for regulating the activity of the shoot meristem in a non-autonomous fashion.     

Genetic interactions reveal the antagonistic roles of FT/TSF and TFL1 in the determination of inflorescence meristem identity in Arabidopsis

During the transition to the reproductive phase, the shoot apical meristem switches from the developmental program that generates vegetative organs to instead produce flowers. In this study, we examined the genetic interactions of FLOWERING LOCUS T (FT)/TWIN SISTER OF FT (TSF) and TERMINAL FLOWER 1 (TFL1) in the determination of inflorescence meristem identity in Arabidopsis thaliana. The ft‐10 tsf‐1 mutants produced a compact inflorescence surrounded by serrated leaves (hyper‐vegetative shoot) at the early bolting stage, as did plants overexpressing TFL1. Plants overexpressing FT or TSF (or both FT and TFL1) generated a terminal flower, as did tfl1‐20 mutants. The terminal flower formed in tfl1‐20 mutants converted to a hyper‐vegetative shoot in ft‐10 tsf‐1 mutants. Grafting ft‐10 tsf‐1 or ft‐10 tsf‐1 tfl1‐20 mutant scions to 35S::FT rootstock plants produced a normal inflorescence and a terminal flower in the scion plants, respectively, although both scions showed similar early flowering. Misexpression of FT in the vasculature and in the shoot apex in wild‐type plants generated a normal inflorescence and a terminal flower, respectively. By contrast, in ft‐10 tsf‐1 mutants the vasculature‐specific misexpression of FT converted the hyper‐vegetative shoot to a normal inflorescence, and in the ft‐10 tsf‐1 tfl1‐20 mutants converted the shoot to a terminal flower. TFL1 levels did not affect the inflorescence morphology caused by FT/TSF overexpression at the early bolting stage. Taking these results together, we proposed that FT/TSF and TFL1 play antagonistic roles in the determination of inflorescence meristem identity, and that FT/TSF are more important than TFL1 in this process.     

Organ boundary NAC‐domain transcription factors are implicated in the evolution of petal fusion

• Research rationale: Evolution of fused petals (sympetaly) is considered to be an important innovation that has repeatedly led to increased pollination efficiency, resulting in accelerated rates of plant diversification. Although little is known about the underlying regulation of sympetaly, genetic pathways ancestrally involved in organ boundary establishment (e.g. CUP SHAPED COTYLEDON [CUC] 1–3 genes) are strong candidates. In sympetalous petunia, mutations in the CUC1/2‐like orthologue NO APICAL MERISTEM (NAM) inhibit shoot apical meristem formation. Despite this, occasional ‘escape shoots’ develop flowers with extra petals and fused inter‐floral whorl organs.
• Central methods: To To determine if petunia CUC‐like genes regulate additional floral patterning, we used virus‐induced silencing (VIGS) following establishment of healthy shoot apices to re‐examine the role of NAM in petunia petal development, and uniquely characterise the CUC3 orthologue NH16.
• Key results: Confirming previous results, we found that reduced floral NAM/NH16 expression caused increased petal–stamen and stamen–carpel fusion, and often produced extra petals. However, further to previous results, all VIGS plants infected with NAM or NH16 constructs exhibited reduced fusion in the petal whorl compared to control plants.
• Main conclusions: Together with previous data, our results demonstrate conservation of petunia CUC‐like genes in establishing inter‐floral whorl organ boundaries, as well as functional evolution to affect the fusion of petunia petals.

     

Dysfunctional mitochondria regulate the size of root apical meristem and leaf development in Arabidopsis

Mitochondria play an important role in maintaining metabolic and energy homeostasis in the plant cell. Thus, perturbation of mitochondrial structure and function will affect plant growth and development. Arabidopsis slow growth3 (slo3) is defective in At3g61360 that encodes a pentatricopeptide repeat (PPR) protein. Analysis of slo3 mitochondrial RNA metabolism revealed that the splicing of nad7 intron 2 is impaired, which leads to a dramatic reduction in complex I activity. So the SLO3 PPR protein is a splicing factor that is required for the removal of nad7 intron 2 in Arabidopsis. The slo3 mutant plants have obvious phenotypes with severe growth retardation and delayed development. The size of root apical meristem (RAM) is reduced and the production of meristem cells is decreased in slo3. Furthermore, the rosette leaves of slo3 are curled or crinkled, which may be derived from uneven growth of the leaf surface. The underlying mechanisms by which dysfunctional mitochondria affect these growth and developmental phenotypes have yet to be established. Nonetheless, plant hormone auxin is known to play an important role in orchestrating the development of RAM and leaf shape. It is possible that dysfunctional mitochondria may interact with auxin signaling pathways to regulate the boundary of RAM and the cell division arrest front during leaf growth in Arabidopsis.     

Expression of CDC2Zm and KNOTTED1 during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation in maize (Zea mays L.) and barley (Hordeum vulgare L.)

Expression of CDC2Zm and KNOTTED1 (KN1) in maize (Zea mays L.) and their cross-reacting proteins in barley (Hordeum vulgare L.) was studied using immunolocalization during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation. Expression of CDC2Zm, a protein involved in cell division, roughly correlated with in-vitro cell proliferation and in the meristematic domes CDC2Zm expression was triggered during in-vitro proliferation. Analysis of the expression of KN1, a protein necessary for maintenance of the shoot meristem, showed that KN1 or KN1-homologue(s) expression was retained in meristematic cells during in-vitro proliferation of axillary shoot meristems. Multiple adventitious shoot meristems appeared to form directly from the KN1- or KN1 homologue(s)-expressing meristematic cells in the in-vitro proliferating meristematic domes. However, unlike Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) leaves ectopically expressing KN1 (G. Chuck et al., 1996 Plant Cell 8: 1277–1289; N. Sinha et al., 1993 Genes Dev. 7: 787–797), transgenic maize leaves over-expressing KN1 were unable to initiate adventitious shoot meristems on their surfaces either in planta or in vitro. Therefore, expression of KN1 is not the sole triggering factor responsible for inducing adventitious shoot meristem formation from in-vitro proliferating axillary shoot meristems in maize. Our results show that genes critical to cell division and plant development have utility in defining in-vitro plant morphogenesis at the molecular level and, in combination with transformation technologies, will be powerful tools in identifying the fundamental molecular and-or genetic triggering factor(s) responsible for reprogramming of plant cells during plant morphogenesis in-vitro. Received: 2 June 1997 / Accepted: 21 July 1997     

A model study of the role of proteins CLV1, CLV2, CLV3, and WUS in regulation of the structure of the shoot apical meristem

In order to elucidate the role of proteins CLV1, CLV2, CLV3, and WUS in the mechanism underlying the maintenance of compartmental structure (spatial arrangement of the zones of biosynthesis of marker proteins) of the shoot apical meristem, a model of such mechanism was developed. Computational experiments led to biologically plausible solutions only when synthesis of substance W in a space between the organizing center and meristem apex was limited by the mechanism based on interaction of CLV3 with membrane receptor CLV1/CLV2 and lower boundary of the zone of W synthesis was determined by isoline of the corresponding threshold level of substance Y concentration. The model of the “reaction-diffusion” type formalizing the role proteins CLV1/CLV2, CLV3, and WUS can describe the basis of the mechanism underlying regulation of the compartmental structure of the shoot apical meristem and positioning of the organizing center in a certain site of the cell ensemble of such meristem.     

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.