Yasuko Kaneko

The molecular mechanisms underlying the synthesis of large cell wall polysaccharides in plant cells are not fully understood. Here, we report that 2 atypical endo-β-1,4-mannanases (MANs), which are not secreted and do not degrade glucomannan in the cell wall, play a role in glucomannan synthesis. Among the 6 MANs in Arabidopsis (Arabidopsis thaliana), AtMAN2 and AtMAN5 contain a transmembrane domain at their N-terminal region instead of a signal peptide. Subcellular localization using MAN protein fused to a fluorescent protein demonstrated that AtMAN2 localizes to the endomembrane system, including the Golgi apparatus, in xylem and interfascicular fiber cells. An Arabidopsis man2 man5 double mutant lost 65% of glucomannan in the cell walls of the inflorescence stem. Immunostaining and immunoelectron microscopic observation also revealed that the man2 man5 double mutant loses glucomannan in the cell walls to about the same extent as the csla2 csla9 double mutant, which lacks major glucomannan synthases. Gene complementation experiments showed that the enzymatic activities of AtMAN2 and AtMAN5 are important for the synthesis of cell wall glucomannan. Arabidopsis possesses another atypical MAN, AtMAN6, with an HDEL retention signal at its C-terminus. However, mutation of AtMAN6 did not affect glucomannan content in the cell walls, suggesting distinct functions for these MANs. This study has identified AtMAN2 and AtMAN5 as factors necessary for normal glucomannan synthesis in Arabidopsis, along with GDP-mannose-generating enzymes and CslAs, and suggests that glucomannan hydrolysis by these MANs contributes to maintaining glucomannan synthesis.

2-methylisoborneol (2-MIB) is one of the main cyanobacterial metabolites responsible for unpleasant taste and odor in drinking water. 2-MIB biosynthesis involves a two-step enzymatic reaction catalyzed by enzymes encoded by the mtf and mtc genes. Two homologous genes, cnbA and cnbB, which encode cyclic nucleotide-binding proteins of unknown function are located upstream and downstream of the mtf and mtc genes, respectively. This study investigated the involvement of cnb genes of 2-MIB-producing cyanobacterium Pseudanabaena foetida var. intermedia NIES 512 in 2-MIB synthesis using heterologous gene expression systems. Transgenic Escherichia coli and Synechococcus elongatus PCC 7942 strains expressing the cnbA-mtf-mtc-cnbB gene cluster exhibited a notable intracellular 2-MIB retention which was not shown by the transgenic strains expressing only the mtf-mtc gene unit. Immunoelectron microscopy detected that the Cnb proteins are associated with unknown intracellular compartments in transgenic S. elongatus PCC 7942 cells. This study suggests that 2-MIB-related Cnb proteins are involved in the formation of intracellular compartments, which are likely associated with intracellular 2-MIB retention.

Heterogeneous distribution of PSI and PSII in thick grana in shade chloroplasts is argued to hinder spillover of chlorophyll excitations from PSII to PSI. To examine this dogma, we measured fluorescence induction at 77K at 690 nm (PSII) and 760 nm (mostly PSI) in the leaf discs of Spinacia oleracea, Cucumis sativus and shade tolerant Alocasia odora, grown at high and low light, and quantified their spillover capacities. PSI fluorescence (FI) consists of the intrinsic PSI fluorescence (FI_α) and fluorescence caused by excitations spilt over from PSII (FI_β). When FI and FII parameters between State 1 and State 2, induced by weak far-red and blue light, were compared, PSII maximum fluorescence (FII_m) and FI_β were greater, and FI_α was smaller in State 1 and thereby the spillover ratio, FI_β/(FI_α+FI_β), was greater in State 1. When non-photochemical quenching (NPQ) was induced, the spillover ratio decreased. Since analyses of F_v/F_m spectra tentatively suggested that about 15% of F_m at 760 nm was from PSII, all data were corrected accordingly. Even after the correction, the spillover ratio in FI_m in State 1 ranged from 16 to 28%. The spillover ratios did not greatly differ between the species or growth light levels. Although extensive grana in low light grown plants would suggest that PSII and PSI are too separated for spillover, the ratios of non-appressed thylakoid membranes/total thylakoid membranes in A. odora chloroplasts were little affected by growth light and more than 40%. Abundant non-appressed thylakoids and margins of appressed-thylakoids would enable efficient spillover. (250 words)

The seed coat protects the embryo from severe external environment. Like the aerial plant parts, the exterior surface of the seed coat is surrounded by a cuticle consisting of lipid polyesters and wax. However, the protective role of the cuticle in mature seeds has not been characterized. We herein study the transcriptional regulation of cuticle formation on the seed surface, which influences seed longevity. A chimeric repressor of Arabidopsis thaliana R2R3 MYB transcription factor gene LATE MERISTEM IDENTITY2 ( LMI2) and a Torenia fournieri ortholog ( TfMYBML3 ) was expressed in A. thaliana and T. fournieri , respectively. These lines and A. thaliana LMI2 T-DNA insertion lines were analyzed for cuticle deficiencies. A. thaliana LMI2 mutants and chimeric repressor lines exhibited a similar cuticle deficiency in immature seeds. Transmission electron microscopy observations, polyester composition analyses, and gene expression investigations revealed that LMI2 plays an essential role in cuticle development in the seed coat epidermis by regulating cutin biosynthesis. The lmi2-2 mutant exhibited shorter seed longevity than the wild-type controls in an accelerated aging test. These results indicate that LMI2 is a key regulator of cuticle formation in the seed coat, and is important for maintaining seed longevity. Significance statement: Cuticle is well known to have multiple important roles for surviving in the terrestrial environment, but their specific role on the seed coat and transcriptional regulation have not yet been elucidated. Here we show that LATE MERISTEM IDENTITY 2 , encoding a transcription factor regulating floral meristem transition, is required for seed cuticle formation and maintaining seed longevity, providing evidence for a link between surface cuticle and seed longevity.

Heterogeneous distribution of PSI and PSII in thick grana in shade chloroplasts is argued to hinder spillover of chlorophyll excitations from PSII to PSI. To examine this dogma, we measured fluorescence induction at 77K at 690 nm (PSII) and 760 nm (mainly PSI) in the leaf discs of Spinacia oleracea , Cucumis sativus and shade tolerant Alocasia odora , grown at high and low light, and quantified their spillover capacities. PSI fluorescence ( F I) consists of the intrinsic PSI fluorescence ( F I _α ) and fluorescence caused by excitations spilt over from PSII ( F I _β ). When F I and F II parameters between State 1 and State 2, induced by weak far-red and blue light, were compared, PSII maximum fluorescence ( F II _m ) and F I _β were greater, and F I _α was smaller in State 1 and thereby the spillover ratio, F I _β /( F I _α +F I _β ) or F I _β / F I _m , was greater in State 1. Since the leftover F II _m was found to be about 10% of total F _m at 760 nm, all the data were corrected. Even after the correction, the spillover ratio in F I _m in State 1 ranged from 21 to 32%, and the spillover ratios were comparable irrespective of growth light conditions. Although extensive grana in low light grown plants would suggest that PSII and PSI are too separated for spillover, in A. odora chloroplasts, the ratio of non-appressed thylakoid membranes/total thylakoid membranes was little affected by growth light and more than 40%. Abundant non-appressed thylakoids would contribute to efficient spillover.

Lipid droplets and membranes in radicle cells from desiccated embryonic axes of soybean (Glycine max) seeds were examined by a recently developed CLEM system, which has been designed to facilitate the observation of identical locations by upright reflected light microscope and compact SEM successively with minimum time lapse. Lipids are major components of membranes and also stored in numerous lipid droplets lining plasma membranes in many seed cells. Fluorescently stained lipid droplets and membranes in the desiccated radicle cells were mainly located along the surface of shrunk protoplasm and around presumptive protein bodies which will turn into vacuoles and increase their volume for radicle protrusion. Co-localization of lipid droplets and membranes suggests the presence of a membrane protection mechanism during desiccation and rehydration processes that ensures prompt elongation of radicle cells during germination.

Desiccation tolerance allows plant seeds to remain viable during desiccation and subsequent re-hydration. In this study, we tried to develop an experimental system to understand the difference between desiccation tolerant and desiccation sensitive radicle cells by examining excised embryonic axes after re-desiccation and subsequent imbibition under various regimes. Embryonic axes excised from soybean (Glycine max (L.) Merr.) seeds imbibed for 3 h to 15 h which remained attached to the cotyledons during imbibition would grow normally after 24 h of desiccation and re-imbibition on wet filter paper. By contrast, when the embryonic axes excised after 3 h imbibition of seeds were kept on wet filter paper for 12 h to 16 h, their growth was significantly retarded after 24 h of desiccation and subsequent re-imbibition. Numerous lipid droplets were observed lining the plasma membrane and tonoplasts in radicle cells of desiccation tolerant embryonic axes before and after desiccation treatment. By contrast, the lipid droplets lining the plasma membrane and tonoplasts became very sparse in radicle cells that were placed for longer times on wet filter paper before desiccation. We observed a clear correlation between the amount of lipid droplets lining plasma membranes and the ability to grow after desiccation and re-imbibition of the excised embryonic axes. In addition to the reduction of lipid droplets in the cells, a gradual increase in starch grains was observed. Large starch grains accumulated in the radicle cells of those axes that failed to grow further.

To visualize the fine structure of compacted DNA of Synechococcus elongatus PCC 7942, which appears at a specific time in the regular light/dark cycle prior to cell division, ChromEM with some modifications was applied. After staining DNA with DRAQ5, the cells were fixed and irradiated by red laser in the presence of 3,3'-diaminobenzidine and subsequently fixed with OsO4. A system with He-Ne laser (633 nm) was set up for efficient irradiation of the bacterial cells in aqueous solution. The compacted DNA was visualized by transmission electron microscopy, in ultrathin sections as electron dense staining by osmium black.

The aquatic carnivorous plant Aldrovanda vesiculosa L. is critically endangered worldwide; its peculiar lifestyle raises many questions and poses problems both intriguing on their own and relevant to conservation. While establishing a culture system for its propagation and restoring its natural habitat in Hozoji pond in Saitama, Japan, we conducted ultrastructural observations to examine the various aspects of Aldrovanda’s way of life. Electron microscopic observation in combination with cryo-techniques produced novel information which could not be obtained by other methods. Some of the results are: phosphorous is stored in petiole cells of turions during winter; mucilaginous guides are provided for pollen tubes in parietal placental ovaries; storage of potassium in the vicinity of the midrib of carnivorous leaves may contribute to the rapid closing of the carnivorous leaves; dynamic sequential changes of the ultrastructure of digestive glands are involved in the synthesis and secretion of digestive enzymes, including protease and acid phosphatase. These results should contribute significantly to our understanding of Aldrovanda and the detailed mechanisms of its life.

Novel para-crystalline structures resembling prolamellar bodies in etioplasts were found in the invasion zones of indeterminate root nodules of Vicia faba, which possess persistent meristems and exhibit sequential developmental stages. The para-crystalline structures existed in most cells in the area of the invasion zone and a hexagonal arrangement of tubular membranes was recognized. Extensive membranes, apparently procured from the structures, were often in contact with the bacteria in young infected cells. We propose that the para-crystalline structures serve as a reservoir of membranes for the formation of the numerous symbiosomes that propagate and fill the infected cells, and suggest naming them pro-symbiosome membrane bodies.