Inhibition of Pre-mRNA Splicing Promotes Root Hair Development in Arabidopsis thaliana.

Root hairs protruding from epidermal cells increase the surface area for water absorption and nutrient uptake. Various environmental factors including light, oxygen concentration, carbon dioxide concentration, calcium and mycorrhizal associations promote root hair formation in Arabidopsis thaliana. Light regulates the expression of a large number of genes at the transcriptional and post-transcriptional levels; however, there is little information linking the light response to root hair development. In this study, we describe a novel mutant, light-sensitive root-hair development 1 (lrh1), that displays enhanced root hair development in response to light. Hypocotyl and root elongation was inhibited in the lrh1 mutant, which had a late flowering phenotype. We identified the gene encoding the p14 protein, a putative component of the splicing factor 3b complex essential for pre-mRNA splicing, as being responsible for the lrh1 phenotype. Indeed, regulation of alternative splicing was affected in lrh1 mutants and treatment with a splicing inhibitor mimicked the lrh1 phenotype. Genome-wide alterations in pre-mRNA splicing patterns including differential splicing events of light signaling- and circadian clock-related genes were found in lrh1 as well as a difference in transcriptional regulation of multiple genes including upregulation of essential genes for root hair development. These results suggest that pre-mRNA splicing is the key mechanism regulating root hair development in response to light signals.

Functionally Diversified Members of the MIR165/6 Gene Family Regulate Ovule Morphogenesis in Arabidopsis thaliana.

The ovules of flowering plants consist of a central embryo sac and surrounding layers of the inner and outer integument. As these structural units eventually give rise to the embryo/endosperm and seed coat, respectively, a precisely organized ovule structure is essential for successful fertilization and seed production. In Arabidopsis thaliana, correct ovule patterning depends on the restricted expression of the CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIP III) gene PHABULOSA (PHB) in the apical region of the incipient inner integument, which in turn is regulated via post-transcriptional suppression by miR165 and miR166 (miR165/6) derived from multiple MIR165/6 genes. While a common subset of MIR165/6 genes regulate PHB expression in the root meristem, leaf primordium and embryo, it is unknown whether the same MIR165/6 subset also regulate PHB expression during ovule development. Furthermore, it is unclear where in the ovule primordia miR165/6 are produced. Here, we show that a distinct set of MIR165/6 genes that are highly expressed in the small regions of early ovule primordia restrict the PHB expression domain to promote integument formation. MIR165/6 genes that function in ovule development are phylogenetically distinct from those acting in roots and leaf primordia. Taken together, our data suggest that members of the MIR165/6 gene family are diversified in their expression capacity to establish elaborate PHB expression patterns depending on the developmental context, thereby allowing HD-ZIP III transcription factors to regulate multiple aspects of plant development.

Accumulation of TIP2;2 Aquaporin during Dark Adaptation Is Partially PhyA Dependent in Roots of Arabidopsis Seedlings.

Light regulates the expression and function of aquaporins, which are involved in water and solute transport. In Arabidopsis thaliana, mRNA levels of one of the aquaporin genes, TIP2;2, increase during dark adaptation and decrease under far-red light illumination, but the effects of light at the protein level and on the mechanism of light regulation remain unknown. Numerous studies have described the light regulation of aquaporin genes, but none have identified the regulatory mechanisms behind this regulation via specific photoreceptor signaling. In this paper, we focus on the role of phytochrome A (phyA) signaling in the regulation of the TIP2;2 protein. We generated Arabidopsis transgenic plants expressing a TIP2;2-GFP fusion protein driven by its own promoter, and showed several differences in TIP2;2 behavior between wild type and the phyA mutant. Fluorescence of TIP2;2-GFP protein in the endodermis of roots in the wild-type seedlings increased during dark adaptation, but not in the phyA mutant. The amount of the TIP2;2-GFP protein in wild-type seedlings decreased rapidly under far-red light illumination, and a delay in reduction of TIP2;2-GFP was observed in the phyA mutant. Our results imply that phyA, cooperating with other photoreceptors, modulates the level of TIP2;2 in Arabidopsis roots.

A comprehensive expression analysis of the Arabidopsis MICRORNA165/6 gene family during embryogenesis reveals a conserved role in meristem specification and a non-cell-autonomous function.

One of the most fundamental events in plant ontogeny is the specification of the shoot and root apical meristem (SAM and RAM) in embryogenesis. In Arabidopsis, the restricted expression of class III homeodomain leucine zipper (HD-ZIP III) transcription factors (TFs) at the central-apical domain of early embryos is required for the correct specification of the SAM and RAM. Because the expression of HD-ZIP III TFs is suppressed by microRNA165/166 (miR165/6), elucidation of the sites of miR165/6 production and their activity range is a key to understanding the molecular basis of SAM and RAM specification in embryogenesis. Here, we present a comprehensive reporter analysis of all nine Arabidopsis MICRORNA165/166 (MIR165/6) genes during embryogenesis. We show that five MIR165/6 genes are transcribed in a largely conserved pattern in embryos, with their expression being preferentially focused at the basal-peripheral region of embryos. Our analysis also indicated that MIR165/6 transcription does not depend on SCARECROW (SCR) function in early embryos, in contrast to its requirement in post-embryonic roots. Furthermore, by observing the expression pattern of the miR-resistant PHBmu-GFP (green fluorescent protein) reporter, in either the presence or absence of the MIR165Amu transgene, which targets PHBmu-GFP, we obtained data that indicate a non-cell-autonomous function for miR165 in early embryos. These results suggest that miR165, and possibly miR166 as well, has the capacity to act as a positional cue from the basal-peripheral region of early embryos, and remotely controls SAM and RAM specification with their non-cell-autonomous function.

Diurnal changes in shoot water dynamics are synchronized with hypocotyl elongation in Arabidopsis thaliana.

We recently demonstrated the circadian clock modulated water dynamics in the roots of a small model plant, Arabidopsis thaliana, by the Nuclear Magnetic Resonance (NMR) microimaging technique. Our developed technique was able to visualize the water distribution that depended on differences in the (1)H signal among region in the shoot, such as the shoot apex, the hypocotyl and the root shoot junction. Water content in the shoot increased during periods of light in comparison with dark periods, and continued through the early stage of seedling growth until the dark period. When the water content changed, elongation and/or movement occurred in the hypocotyl, and these events were synchronized. The water dynamics of the shoot also displayed an opposite phase with the root water dynamics.

The circadian clock modulates water dynamics and aquaporin expression in Arabidopsis roots.

We have developed a plant growth system to analyze water dynamics in the roots of a small model plant, Arabidopsis thaliana, by nuclear magnetic resonance (NMR) microscopic imaging. Using the two-dimensional slice technique, we obtained a series of images with high signal-to-noise ratio indicating the water distribution in the root. To demonstrate light regulation of water transport in the root and involvement of aquaporin gene expression, we visualized the distribution of water in Arabidopsis roots under various light conditions and compared the data with the expression profiles of two aquaporin genes. (1)H-NMR imaging revealed that water content in Arabidopsis roots is lower in the light than in the dark. This diurnal variation in water content was clearly observed in the basal zone of the root. In addition, an autonomous rhythm of water dynamics was observed under continuous light (LL) and darkness (DD). However, the circadian oscillation in water dynamics was obscured in the early-flowering 3 (elf3) mutant under LL. The expression of both the aquaporin genes, AtPIP1;2 and AtPIP2;1, oscillated with the circadian rhythm under LL conditions in wild-type seedlings, but not in the elf3 mutant. These results demonstrate the advantages of our technique for monitoring water dynamics in roots of living Arabidopsis seedlings, and suggest that the circadian clock modulates water dynamics and aquaporin expression.

Expression of photosynthesis-related genes and their regulation by light during somatic embryogenesis in Daucus carota.

To clarify the spatial and temporal pattern of gene expression for photosynthesis-associated proteins during somatic embryogenesis in Daucus carota L., the localization of mRNAs for three genes, rbcL, Lhcb and por, was examined in dark-grown and light-irradiated somatic embryos by in situ hybridization. The three mRNAs were expressed in common in the mesophyll precursor cells of light-irradiated embryos at the late torpedo and plantlet stages, but characteristic expression patterns of each photosynthesis-related gene were also observed. Expression of rbcL mRNA first occurred throughout the embryo but gradually became localized in the mesophyll precursor cells and cortex during early embryogenesis. Localization of Lhcb mRNA in the mesophyll precursor cells and shoot apical meristem became clear in the early torpedo stage. Expression of Lhcb mRNA was not affected by light during early embryogenesis, but could be induced by light in the torpedo stage, suggesting that light-inducible expression of Lhcb mRNA arises within the torpedo stage. At the late torpedo stage, clear localization of por mRNA started in mesophyll precursor cells of the cotyledon in light-irradiated embryos. Greening potency of the embryo also appeared first at this stage. Therefore, greening and initial differentiation of photosynthetic tissues during somatic embryogenesis seem to be associated with coordinated expression of mRNA for rbcL, Lhcb and por in late torpedo-shaped embryos.

Sugar-induced adventitious roots in Arabidopsis seedlings.

The effects of sugars on root growth and on development of adventitious roots were analyzed in Arabidopsis thaliana. Seeds were sown on agar plates containing 0.0-5.0% sugars and placed vertically in darkness (DD) or under long day (LD, 16 h:8 h) conditions, so that the seedlings were constantly attached to the agar medium. In the sucrose-supplemented medium, seedlings showed sustained growth in both DD and LD. However, only dark-grown seedlings developed adventitious roots from the elongated hypocotyl. The adventitious roots began to develop 5 days after imbibition and increased in number until day 11. They could, however, be initiated at any position along the hypocotyl, near the cotyledon or the primary root. They were initiated in the pericycle in the same manner as ordinary lateral roots. Sucrose, glucose and fructose greatly stimulated the induction of adventitious roots, but mannose or sorbitol did not. Sucrose at concentrations of 0.5-2.0% was most effective in inducing adventitious roots, although 5.0% sucrose suppressed induction. Direct contact of the hypocotyl with the sugar-supplemented agar medium was indispensable for the induction of adventitious roots.

Detection of ethylene receptor protein Cm-ERS1 during fruit development in melon (Cucumis melo L.).

Antibodies against melon ethylene receptor, Cm- ERS1 was prepared. Cm-ERS1 protein formed a disulphide-linked homodimer and it was present in microsomal membranes but not in soluble fractions. Cm-ERS1 protein was present at high levels in melon fruit during early developmental stages. This transition pattern was also observed in another melon cultivar.