The axial body pattern of Arabidopsis is determined during embryogenesis by auxin signaling and differential gene expression. Here we demonstrate that another pathway, cell-to-cell communication through plasmodesmata (PD), is regulated during apicalbasal pattern formation. The SHOOT MERISTEMLESS (STM) promoter was used to drive expression in the shoot apical meristem (SAM) and a subset of cells at the base of the hypocotyl of 1؋, 2؋, and 3؋ soluble green fluorescent proteins (sGFPs), and the P30 movement protein of Tobacco mosaic virus (TMV) translationally fused to 1؋ and 2؋ sGFP. In the early heart stage, 2؋ sGFP (54 kDa) moves throughout the whole embryo, whereas 3؋ sGFP (81 kDa) shows more restricted movement. As the embryo develops, PD apertures are down regulated to form local subdomains allowing transport of different sized tracers. For example, movement of 2؋ sGFP to the cotyledon, and 3؋ sGFP to root tips, becomes restricted. Subdomains of cell-to-cell transport align with the apical-basal embryo body axis and correspond to the shoot apex, cotyledons, hypocotyl, and root. Studies with P30 -GFP fusions reinforce the distinction between embryonic symplastic subdomains. Although P30 targets embryo cell walls as puncta (diagnostic for functional localization of P30 to PD in adult plants), P30 cannot dilate embryonic PD to overcome the barriers for transport between symplastic subdomains, suggesting that specific boundaries separate symplastic subdomains of the embryo. Thus, cell-to-cell communication via plasmodesmata conveys positional information critical to establish the axial body pattern during embryogenesis in Arabidopsis.GFP ͉ symplast ͉ Tobacco mosaic virus movement protein P30 ͉ STM

There is increasing evidence for intercellular trafficking of macromolecules through plasmodesmata (PD) during plant development. Here we study the ability of PD to traffic proteins during embryogenesis and early seedling development in Arabidopsis. Transgenic lines that induce GFP expression only in meristems, MSG (meristem-specific GFP), were used to monitor GFP movement. Cell-to-cell movement of different-sized GFP reporters reveals that embryos and young seedlings traffic proteins at least 54 kDa in size. Although 27-kDa soluble GFP (1؋sGFP) freely moves between cells throughout the entire embryo during all stages analyzed, 2؋sGFP movement becomes more restricted as development proceeds. After germination, cells near the apical meristem in seedlings show a higher size exclusion limit (SEL), whereas the SEL becomes more restricted as surrounding tissues develop identities. Although 1؋sGFP moves throughout leaf primordia, as the leaf develops only the basal part of leaf petioles, main vascular tissues, and leaf veins (not blades) allow 1؋sGFP movement. Although previous studies showed that embryos allow movement of small symplastic tracers (0.5 kDa), the present data demonstrate that the embryo constitutes a single symplast that allows transport of macromolecules as well. Even 2؋sGFP moves from its site of expression at the apical meristem in embryos and seedlings, yet the extent of movement is more limited than 1؋sGFP. Thus, PD have distinct SELs in different subregions of the embryo and seedling. These studies support the general concept that PD in younger tissues are more dilated and less restrictive than PD in older (nonvascular) tissues.plasmodesmata ͉ symplast

We identify a gene, ORGAN BOUNDARY1 (OBO1), by its unique pattern of enhancer-driven GFP expression at the boundaries between the apical meristems and lateral organs in Arabidopsis embryos, seedlings, and mature plants. OBO1 also is expressed at the root apical meristem and in distinct cell files surrounding this area. OBO1 is one of a 10-member plant-specific gene family encoding a single small domain (133 amino acids) with unknown function. One member of this gene family, OBO2, is identical to a previously studied gene, LIGHT-SENSITIVE HYPOCOTYL1. Overexpression of OBO1 causes an abnormal number and size of petals and petalstamen fusions. The patterns of OBO1 gene expression are distinct but overlap with other genes involved in boundary formation in the Arabidopsis shoot apical meristem, including CUP-SHAPED COTYLEDON, LATERAL ORGAN BOUNDARIES, BLADE-ON-PETIOLE, ASYMMETRIC LEAVES, and LATERAL ORGAN FUSION. Nuclear localization of OBO1 suggests that it might act with one or more of the transcription factors encoded by the foregoing genes. Ablation of the specific cells expressing OBO1 leads to loss of the shoot apical meristem and lateral organs. Thus, the cells expressing OBO1 are important for meristem maintenance and organogenesis in Arabidopsis.

In plants, plasmodesmata (PD) serve as channels for micromolecular and macromolecular cell-to-cell transport. Based on structure, PD in immature tissues are classified into two types, simple and branched (X-and Y-shaped) or twinned. The maximum size of molecules capable of PD transport defines PD aperture, known as the PD size exclusion limit. Here we report an Arabidopsis mutation, decreased size exclusion limit1 (dse1), that exhibits reduced cell-to-cell transport of the small (524 Da) fluorescent tracer 8-hydroxypyrene-1,3,6-trisulfonic acid at the midtorpedo stage of embryogenesis. Correspondingly, the fraction of X-and Y-shaped and twinned PD was reduced in dse1 embryos compared with WT embryos at this stage, suggesting that the frequency of PD is related to transport capability. dse1 is caused by a point mutation in At4g29860 (previously termed TANMEI) at the last donor splice site of its transcript, resulting in alternative splicing in both the first intron and the last intron. AtDSE1 is a conserved eukaryotic 386-aa WD-repeat protein critical for Arabidopsis morphogenesis and reproduction. Similar to its homologs in mouse, null mutants are embryo-lethal. The weak loss-of-function mutant dse1 exhibits pleiotropic phenotypes, including retarded vegetative growth, delayed flowering time, dysfunctional male and female organs, and delayed senescence. Finally, silencing of DSE1 in Nicotiana benthamiana leaves leads to reduced movement of GFP fused to tobacco mosaic virus movement protein. Thus, DSE1 is important for regulating PD transport between plant cells.

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