Tomoo Shimada

Stomagen positively regulates stomatal density in Arabidopsis.

Stomata in the epidermal tissues of leaves are valves through which passes CO2, and as such they influence the global carbon cycle. The two-dimensional pattern and density of stomata in the leaf epidermis are genetically and environmentally regulated to optimize gas exchange. Two putative intercellular signalling factors, EPF1 and EPF2, function as negative regulators of stomatal development in Arabidopsis, possibly by interacting with the receptor-like protein TMM. One or more positive intercellular signalling factors are assumed to be involved in stomatal development, but their identities are unknown. Here we show that a novel secretory peptide, which we designate as stomagen, is a positive intercellular signalling factor that is conserved among vascular plants. Stomagen is a 45-amino--rich peptide that is generated from a 102-amino-acid precursor protein designated as STOMAGEN. Both an in planta analysis and a semi-in-vitro analysis with recombinant and chemically synthesized stomagen peptides showed that stomagen has stomata-inducing activity in a dose-dependent manner. A genetic analysis showed that TMM is epistatic to STOMAGEN (At4g12970), suggesting that stomatal development is finely regulated by competitive binding of positive and negative regulators to the same receptor. Notably, STOMAGEN is expressed in inner tissues (the mesophyll) of immature leaves but not in the epidermal tissues where stomata develop. This study provides evidence of a mesophyll-derived positive regulator of stomatal density. Our findings provide a conceptual advancement in understanding stomatal development: inner photosynthetic tissues optimize their function by regulating stomatal density in the epidermis for efficient uptake of CO2.

Vacuolar sorting receptor for seed storage proteins in Arabidopsis thaliana

The seeds of higher plants accumulate large quantities of storage protein. During seed maturation, storage protein precursors synthesized on rough endoplasmic reticulum are sorted to protein storage vacuoles, where they are converted into the mature forms and accumulated. Previous attempts to determine the sorting machinery for storage proteins have not been successful. Here we show that a type I membrane protein, AtVSR1/AtELP, of Arabidopsis functions as a sorting receptor for storage proteins. The atvsr1 mutant missorts storage proteins by secreting them from cells, resulting in an enlarged and electron-dense extracellular space in the seeds. The atvsr1 seeds have distorted cells and smaller protein storage vacuoles than do WT seeds, and atvsr1 seeds abnormally accumulate the precursors of two major storage proteins, 12S globulin and 2S albumin, together with the mature forms of these proteins. AtVSR1 was found to bind to the C-terminal peptide of 12S globulin in a Ca2+-dependent manner. These findings demonstrate a receptor-mediated transport of seed storage proteins to protein storage vacuoles in higher plants.

Myosin-dependent endoplasmic reticulum motility and F-actin organization in plant cells.

Plants exhibit an ultimate case of the intracellular motility involving rapid organelle trafficking and continuous streaming of the endoplasmic reticulum (ER). Although it was long assumed that the ER dynamics is actomyosin-driven, the responsible myosins were not identified, and the ER streaming was not characterized quantitatively. Here we developed software to generate a detailed velocity-distribution map for the GFP-labeled ER. This map revealed that the ER in the most peripheral plane was relatively static, whereas the ER in the inner plane was rapidly streaming with the velocities of up to ∼3.5 μm/sec. Similar patterns were observed when the cytosolic GFP was used to evaluate the cytoplasmic streaming. Using gene knockouts, we demonstrate that the ER dynamics is driven primarily by the ER-associated myosin XI-K, a member of a plant-specific myosin class XI. Furthermore, we show that the myosin XI deficiency affects organization of the ER network and orientation of the actin filament bundles. Collectively, our findings suggest a model whereby dynamic three-way interactions between ER, F-actin, and myosins determine the architecture and movement patterns of the ER strands, and cause cytosol hauling traditionally defined as cytoplasmic streaming.

A novel role for oleosins in freezing tolerance of oilseeds in Arabidopsis thaliana

SUMMARY Oil bodies in seeds of higher plants are surrounded with oleosins. Here we demonstrate a novel role for oleosins in protecting oilseeds against freeze/thaw-induced damage of their cells. We detected four oleosins in oil bodies isolated from seeds of Arabidopsis thaliana, and designated them OLE1, OLE2, OLE3 and OLE4 in decreasing order of abundance in the seeds. For reverse genetics, we isolated oleosin-deficient mutants (ole1, ole2, ole3 and ole4) and generated three double mutants (ole1 ole2, ole1 ole3 and ole2 ole3). Electron microscopy showed an inverse relationship between oil body sizes and total oleosin levels. The double mutant ole1 ole2, which had the lowest levels of oleosins, had irregular enlarged oil-containing structures throughout the seed cells. Germination rates were positively associated with oleosin levels, suggesting that defects in germination are related to the expansion of oil bodies due to oleosin deficiency. We found that freezing followed by imbibition at 4 degrees C abolished seed germination of single mutants (ole1, ole2 and ole3), which germinated normally without freezing treatment. The treatment accelerated the fusion of oil bodies and the abnormal-positioning and deformation of nuclei in ole1 seeds, which caused seed mortality. In contrast, ole1 seeds that had undergone freezing treatment germinated normally when incubated at 22 degrees C instead of 4 degrees C, because degradation of oils abolished the acceleration of fusion of oil bodies during imbibition. Taken together, our findings suggest that oleosins increase the viability of over-wintering oilseeds by preventing abnormal fusion of oil bodies during imbibition in the spring.

CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L.

Targeted genome modification technologies are key tools for functional genomics. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 system (CRISPR/Cas9) is an emerging technology for targeted genome modification. The CRISPR/Cas9 system consists of a short guide RNA (gRNA), which specifies the target genome sequence, and the Cas9 protein, which has endonuclease activity. The CRISPR/Cas9 system has been applied to model animals and flowering plants, including rice, sorghum, wheat, tobacco and Arabidopsis. Here, we report the application of CRISPR/Cas9 to targeted mutagenesis in the liverwort Marchantia polymorpha L., which has emerged as a model species for studying land plant evolution. The U6 promoter of M. polymorpha was identified and cloned to express the gRNA. The target sequence of the gRNA was designed to disrupt the gene encoding auxin response factor 1 (ARF1) in M. polymorpha. Using Agrobacterium-mediated transformation, we isolated stable mutants in the gametophyte generation of M. polymorpha. CRISPR/Cas9-based site-directed mutagenesis in vivo was achieved using either the Cauliflower mosaic virus 35S or M. polymorpha EF1α promoter to express Cas9. Isolated mutant individuals showing an auxin-resistant phenotype were not chimeric. Moreover, stable mutants were produced by asexual reproduction of T1 plants. Multiple arf1 alleles were easily established using CRIPSR/Cas9-based targeted mutagenesis. Our results provide a rapid and simple approach for molecular genetics in M. polymorpha, and raise the possibility that CRISPR/Cas9 may be applied to a wide variety of plant species.

A rapid and non-destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana

The creation of transgenic plants has contributed extensively to the advancement of plant science. Establishing homozygous transgenic lines is time-consuming and laborious, and using antibiotics or herbicides to select transformed plants may adversely affect the growth of some transgenic plants. Here we describe a novel technology, which we have named FAST (fluorescence-accumulating seed technology), that overcomes these difficulties. Although this technology was designed for use in Arabidopsis thaliana, it may be adapted for use in other plants. The technology is based on the expression of a fluorescent co-dominant screenable marker FAST, under the control of a seed-specific promoter, on the oil body membrane. The FAST marker harbors a fusion gene encoding either GFP or RFP with an oil body membrane protein that is prominent in seeds. The marker protein was only expressed in a specific organ (i.e. in dry seeds) and at a specific time (i.e. during dormancy), which are desirable features of selectable and/or screenable markers. This technique provides an immediate and non-destructive method for identifying transformed dry seeds. It identified the heterozygous transformed seeds among the T(1) population and the homozygous seeds among the T(2) population with a false-discovery rate of <1%. The FAST marker reduces the length of time required to produce homozygous transgenic lines from 7.5 to 4 months. Furthermore, it does not require sterilization, clean-bench protocols or the handling of large numbers of plants. This technology should greatly facilitate the generation of transgenic Arabidopsis plants.

KATAMARI1/MURUS3 Is a Novel Golgi Membrane Protein That Is Required for Endomembrane Organization in Arabidopsis

In plant cells, unlike animal and yeast cells, endomembrane dynamics appear to depend more on actin filaments than on microtubules. However, the molecular mechanisms of endomembrane–actin filament interactions are unknown. In this study, we isolated and characterized an Arabidopsis thaliana mutant, katamari1 (kam1), which has a defect in the organization of endomembranes and actin filaments. The kam1 plants form abnormally large aggregates that consist of endoplasmic reticulum with actin filaments in the perinuclear region within the cells and are defective in normal cell elongation. Map-based cloning revealed that the KAM1 gene is allelic to the MUR3 gene. We demonstrate that the KAM1/MUR3 protein is a type II membrane protein composed of a short cytosolic N-terminal domain and a transmembrane domain followed by a large lumenal domain and is localized specifically on Golgi membranes. We further show that actin filaments interact with Golgi stacks via KAM1/MUR3 to maintain the proper organization of endomembranes. Our results provide functional evidence that KAM1/MUR3 is a novel component of the Golgi-mediated organization of actin functioning in proper endomembrane organization and cell elongation.

Arabidopsis VPS35, a retromer component, is required for vacuolar protein sorting and involved in plant growth and leaf senescence.

The retromer complex is responsible for retrograde transport, which is coordinated with anterograde transport in the secretory pathway including vacuolar protein sorting. Yeast VPS35 is a component of the retromer complex that is essential for recognition of specific cargo molecules. The physiological function of VPS35 has not been determined in vacuolar protein sorting in higher organisms. Arabidopsis thaliana has three VPS35 homologs designated VPS35a, VPS35b and VPS35c. We isolated four vps35 mutants (vps35a-1, vps35b-1, vps35b-2 and vps35c-1) and then generated four double mutants and one triple mutant. vps35a-1 vps35c-1 exhibited no unusual phenotypes. On the other hand, vps35b-1 vps35c-1 and the triple mutant (vps35a-1 vps35b-2 vps35c-1) exhibited severe phenotypes: dwarfism, early leaf senescence and fragmentation of protein storage vacuoles (PSVs). In addition, these mutants mis-sorted storage proteins by secreting them out of the cells and accumulated a higher level of vacuolar sorting receptor (VSR) than the wild type. VPS35 was localized in pre-vacuolar compartments (PVCs), some of which contained VSR. VPS35 was immunoprecipitated with VPS29/MAG1, another component of the retromer complex. Our findings suggest that VPS35, mainly VPS35b, is involved in sorting proteins to PSVs in seeds, possibly by recycling VSR from PVCs to the Golgi complex, and is also involved in plant growth and senescence in vegetative organs.

An Endoplasmic Reticulum-Derived Structure That Is Induced under Stress Conditions in Arabidopsis

The endoplasmic reticulum (ER) body is a characteristic structure derived from ER and is referred to as a proteinase-sorting system that assists the plant cell under various stress conditions. Fluorescent ER bodies were observed in transgenic plants of Arabidopsis expressing green fluorescent protein fused with an ER retention signal. ER bodies were widely distributed in the epidermal cells of whole seedlings. In contrast, rosette leaves had no ER bodies. We found that wound stress induced the formation of many ER bodies in rosette leaves. ER bodies were also induced by treatment with methyl jasmonate (MeJA), a plant hormone involved in the defense against wounding and chewing by insects. The induction of ER bodies was suppressed by ethylene. An electron microscopic analysis showed that typical ER bodies were induced in the non-transgenic rosette leaves treated with MeJA. An experiment using coi1 and etr1-4 mutant plants showed that the induction of ER bodies was strictly coupled with the signal transduction of MeJA and ethylene. These results suggested that the formation of ER bodies is a novel and unique type of endomembrane system in the response of plant cells to environmental stresses. It is possible that the biological function of ER bodies is related to defense systems in higher plants.

Enhancement of leaf photosynthetic capacity through increased stomatal density in Arabidopsis

Photosynthetic rate is determined by CO2 fixation and CO2 entry into the plant through pores in the leaf epidermis called stomata. However, the effect of increased stomatal density on photosynthetic rate remains unclear. This work investigated the effect of alteration of stomatal density on leaf photosynthetic capacity in Arabidopsis thaliana. Stomatal density was modulated by overexpressing or silencing STOMAGEN, a positive regulator of stomatal development. Leaf photosynthetic capacity and plant growth were examined in transgenic plants. Increased stomatal density in STOMAGEN-overexpressing plants enhanced the photosynthetic rate by 30% compared to wild-type plants. Transgenic plants showed increased stomatal conductance under ambient CO2 conditions and did not show alterations in the maximum rate of carboxylation, indicating that the enhancement of photosynthetic rate was caused by gas diffusion changes. A leaf photosynthesis-intercellular CO2 concentration response curve showed that photosynthetic rate was increased under high CO2 conditions in association with increased stomatal density. STOMAGEN overexpression did not alter whole plant biomass, whereas its silencing caused biomass reduction. Our results indicate that increased stomatal density enhanced leaf photosynthetic capacity by modulating gas diffusion. Stomatal density may be a target trait for plant engineering to improve photosynthetic capacity.