Autonomous differentiation of transgenic cells requiring no external hormone application: the endogenous gene expression and phytohormone behaviors.

The ectopic overexpression of developmental regulator (DR) genes has been reported to improve the transformation in recalcitrant plant species because of the promotion of cellular differentiation during cell culture processes. In other words, the external plant growth regulator (PGR) application during the tissue and cell culture process is still required in cases utilizing DR genes for plant regeneration. Here, the effect of Arabidopsis BABY BOOM (BBM) and WUSCHEL (WUS) on the differentiation of tobacco transgenic cells was examined. We found that the SRDX fusion to WUS, when co-expressed with the BBM-VP16 fusion gene, significantly influenced the induction of autonomous differentiation under PGR-free culture conditions, with similar effects in some other plant species. Furthermore, to understand the endogenous background underlying cell differentiation toward regeneration, phytohormone and RNA-seq analyses were performed using tobacco leaf explants in which transgenic cells were autonomously differentiating. The levels of active auxins, cytokinins, abscisic acid, and inactive gibberellins increased as cell differentiation proceeded toward organogenesis. Gene Ontology terms related to phytohormones and organogenesis were identified as differentially expressed genes, in addition to those related to polysaccharide and nitrate metabolism. The qRT-PCR four selected genes as DEGs supported the RNA-seq data. This differentiation induction system and the reported phytohormone and transcript profiles provide a foundation for the development of PGR-free tissue cultures of various plant species, facilitating future biotechnological breeding.

Behavior of Male Gamete Fusogen GCS1/HAP2 and the Regulation in Arabidopsis Double Fertilization.

In the sexual reproduction of flowering plants, two independent fertilization events occur almost simultaneously: two identical sperm cells fuse with either the egg cell or the central cell, resulting in embryo and endosperm development to produce a seed. GCS1/HAP2 is a sperm cell membrane protein essential for plasma membrane fusion with both female gametes. Other sperm membrane proteins, DMP8 and DMP9, are more important for egg cell fertilization than that of the central cell, suggesting its regulatory mechanism in GCS1/HAP2-driving gamete membrane fusion. To assess the GCS1/HAP2 regulatory cascade in the double fertilization system of flowering plants, we produced Arabidopsis transgenic lines expressing different GCS1/HAP2 variants and evaluated the fertilization in vivo. The fertilization pattern observed in GCS1_RNAi transgenic plants implied that sperm cells over the amount of GCS1/HAP2 required for fusion on their surface could facilitate membrane fusion with both female gametes. The cytological analysis of the dmp8dmp9 sperm cell arrested alone in an embryo sac supported GCS1/HAP2 distribution on the sperm surface. Furthermore, the fertilization failures with both female gametes were caused by GCS1/HAP2 secretion from the egg cell. These results provided a possible scenario of GCS1/HAP2 regulation, showing a potential scheme for capturing additional GCS1/HAP2-interacting proteins.

Development of an inducible excision system of a visual marker Ipomoea batatas Myb gene from the genome of transgenic cells.

In the plant genetic transformation process, single selection by a chemical-resistant marker gene occasionally allows the proliferation of non-transgenic cells, escaping selection pressure. The additional use of a visual marker gene is effective for accurate selection. For instance, R2R3-MYB genes are used for regulating anthocyanin biosynthesis; however, constitutive Myb expression in transgenic plants is not always desirable and may cause developmental abnormalities due to excess anthocyanin accumulation. To overcome the remaining problems in the use of Myb as a visible marker, we developed T-DNA. Ipomoea batatas Myb (IbMyb) and Cre expression cassettes were inserted between two loxP sequences, and the hygromycin phosphotransferase (HPT) and green fluorescent protein (GFP) expression cassettes were located outside of the loxP-IbMyb-Cre-loxP region. In the developed system, IbMyb and Cre were excised from the genomes of transgenic cells using heat-inducible Cre-loxP recombination. Upon heat treatment in a general incubator, green shoots emerged from purple tobacco transgenic calli that were pigmented with IbMyb expression. The excision of IbMyb from the genome of green shoots was confirmed using polymerase chain reaction (PCR) and sequencing. GFP expression was observed in the roots of the obtained green transgenic plants. We report that the system developed here operated successfully in tobacco, showing the potential to provide an easier and cheaper visual selection of transgenic cells in the genetic transformation process.

Restricted Pollination for Tracing Individual Pollen Tubes in a Pistil.

As one of the essential steps to complete sexual reproduction, a pollen tube is precisely guided to an embryo sac to deliver the sperm cells. This ovule targeting by a pollen tube is one of the essential steps in pollen tube guidance. To assess the ovule targeting ability of the pollen tube from a certain mutant line, comparative analysis of pollen tube behaviors between wild-type and mutant genotypes is important. Here, we provide a protocol that traces all pollen tubes germinated from the quartet tetrad in a pistil by restricted pollination and aniline blue staining. By this analysis, statistical comparison between wild-type and the mutant pollen tube functions under the same in vivo condition is possible.

Behavior of filamentous temperature-sensitive Z2 (FtsZ2) in the male gametophyte during sexual reproduction processes of flowering plants.

Filamentous temperature-sensitive Z (FtsZ) is a critical division protein in bacteria that functions in forming a Z-ring structure to constrict the cell. Since the establishment of the plastid by endosymbiosis of a cyanobacterium into a eukaryotic cell, division via Z-ring formation has been conserved in the plastids of flowering plants. The FtsZ gene was transferred from the cyanobacterial ancestor of plastids to the eukaryotic nuclear genome during evolution, and flowering plants evolved two FtsZ homologs, FtsZ1 and FtsZ2, which are involved in chloroplast division through distinct molecular functions. Regarding the behaviors of FtsZ in nonphotosynthetic cells, the plastid localization of FtsZ1 proteins in the cytoplasm of microspores and pollen vegetative cells but not in generative cells or sperm cells has been reported. On the other hand, the significant accumulation of FtsZ2 transcripts in generative cells has been reported. However, the synthesis of FtsZ2 in the male gamete has not been investigated. Additionally, FtsZ2 behavior has not been analyzed in pollen, a nonphotosynthetic male tissue. Here, we report FtsZ2 protein behaviors in the male gamete by analyzing the localization patterns of GFP-fused protein at various pollen developmental stages and in gametes during the fertilization process. Our results showed that FtsZ2 localization coincided with that of plastids. FtsZ2 protein in male gametes was almost absent, despite the presence of the transcripts. Moreover, transmission of paternal FtsZ2 transcripts to the zygote and endosperm was not observed.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization.

Flowering plants have a unique sexual reproduction system called 'double fertilization', in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes' nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

The male gamete membrane protein DMP9/DAU2 is required for double fertilization in flowering plants.

All flowering plants exhibit a unique type of sexual reproduction called 'double fertilization' in which each pollen tube-delivered sperm cell fuses with an egg and a central cell. Proteins that localize to the plasma membrane of gametes regulate one-to-one gamete pairing and fusion between male and female gametes for successful double fertilization. Here, we have identified a membrane protein from Lilium longiflorum generative cells using proteomic analysis and have found that the protein is an ortholog of Arabidopsis DUF679 DOMAIN MEMBRANE PROTEIN 9 (DMP9)/DUO1-ACTIVATED UNKNOWN 2 (DAU2). The flowering plant DMP9 proteins analyzed in this study were predicted to have four transmembrane domains and be specifically expressed in both generative and sperm cells. Knockdown of DMP9 resulted in aborted seeds due to single fertilization of the central cell. Detailed imaging of DMP9-knockdown sperm cells during in vivo and semi-in vitro double fertilization revealed that DMP9 is involved in gamete interaction that leads to correct double fertilization.

Loss of GCS1/HAP2 does not affect the ovule-targeting behavior of pollen tubes.

KEY MESSAGE : hap2-1 pollen tube ovule targeting. Upon pollination, a pollen grain germinates to produce a pollen tube, which grows through the style to deliver two immobile sperm cells to the female gametophyte. Double fertilization is completed after the pollen tube enters an ovule. GENERATIVE CELL SPECIFIC 1 (GCS1)/HAPLESS 2 (HAP2) contributes to the fusion of gametes at fertilization and has been suggested to affect pollen tube guidance. However, there is controversy over the role of GCS1/HAP2 in pollen tube guidance because of conflicting results from different studies. To characterize the effects of the gcs1/hap2 mutation on pollen tube behavior, we analyzed the Arabidopsis thaliana hap2-1/HAP2 mutant, which carries a gcs1/hap2 mutation in the quartet background. The quartet mutant produces tetrads consisting of four pollen grains that remain adherent after the pollen mother cell has completed meiosis. Thus, a hap2-1/HAP2 tetrad contains hap2-1 and HAP2 pollen grains in a 2:2 ratio. Moreover, the hap2-1 locus is linked to the ß-glucuronidase (GUS) gene. An excess pollination experiment with hap2-1/HAP2 tetrads revealed that the hap2-1 pollen tube targets ovules normally. Additionally, the results of restricted pollination and aniline blue staining indicated that there are no significant differences between the ovule-targeting frequencies of pollen tubes from hap2-1/HAP2 and HAP2/HAP2 tetrads.

Isolation of GFP-tagged plasma membrane protein from Arabidopsis egg cells.

Angiosperms possess a double fertilization system for sexual reproduction. Double fertilization is regulated by interactions among proteins localized in the plasma membrane of each sex gamete. A few plasma membrane resident proteins regulating double fertilization have been identified in male gametes. In contrast, no fertilization regulators in female gamete plasma membrane have been identified, largely due to difficulties in the isolation and collection of female gametes. We had produced Arabidopsis transgenic plant pDD45::GFP-AtPIP2;1 where the egg cell plasma membrane was specifically labeled with GFP (Igawa et al. 2013). The protein extract derived from approximately 200 pistils, which contained unfertilized and mature egg cells, was subjected to immunoprecipitation using anti-GFP antibody. As a result, both GFP and AtPIP2;1 were specifically detected in immunoprecipitated proteins from pistil tissues of pDD45::GFP-AtPIP2;1 transgenic plant, but not in those of wild type pistils. It was revealed that specific proteins expressed in the egg cells were successfully isolated from pistil cell population. The method described here showed the feasibility of isolating specific egg cell plasma membrane protein without gamete isolation and collection procedures.

Gamete Dialogs in Green Lineages.

Gamete fusion is a core process of sexual reproduction and, in both plants and animals, different sex gametes fuse within species. Although most of the molecular factors involved in gamete interaction are still unknown in various sex-possessing eukaryotes, reports of such factors in algae and land plants have been increasing in the past decade. In particular, knowledge of gamete interaction in flowering plants and green algae has increased since the identification of the conserved gamete fusion factor generative cell specific 1/hapless 2 (GCS1/HAP2). GCS1 was first identified as a pollen generative cell-specific transmembrane protein in the lily (Lilium longiflorum), and was then shown to function not only in flowering plant gamete fusion but also in various eukaryotes, including unicellular protists and metazoans. In addition, although initially restricted to Chlamydomonas, knowledge of gamete attachment in flowering plants was also acquired. This review focuses on recent progress in the study of gamete interaction in volvocine green algae and flowering plants and discusses conserved mechanisms of gamete recognition, attachment, and fusion leading to zygote formation.

Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism.

In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization.

Resistance to Sri Lankan cassava mosaic virus (SLCMV) in genetically engineered cassava cv. KU50 through RNA silencing.

Cassava ranks fifth among the starch producing crops of the world, its annual bioethanol yield is higher than for any other crop. Cassava cultivar KU50, the most widely grown cultivar for non-food purposes is susceptible to Sri Lankan cassava mosaic virus (SLCMV). The objective of this work was to engineer resistance to SLCMV by RNA interference (RNAi) in order to increase biomass yield, an important aspect for bioethanol production. Here, we produced transgenic KU50 lines expressing dsRNA homologous to the region between the AV2 and AV1 of DNA A of SLCMV. High level expression of dsRNA of SLCMV did not induce any growth abnormality in the transgenic plants. Transgenic lines displayed high levels of resistance to SLCMV compared to the wild-type plants and no virus load could be detected in uninoculated new leaves of the infected resistant lines after PCR amplification and RT-PCR analysis. The agronomic performance of the transgenic lines was unimpaired after inoculation with the virus as the plants presented similar growth when compared to the mock inoculated control plants and revealed no apparent reduction in the amount and weight of tubers produced. We show that the resistance is correlated with post-transcriptional gene silencing because of the production of transgene specific siRNA. The results demonstrate that transgenic lines exhibited high levels of resistance to SLCMV. This resistance coupled with the desirable yield components in the transgenic lines makes them better candidates for exploitation in the production of biomass as well as bioethanol.

Gamete attachment process revealed in flowering plant fertilization.

Sex-possessing organisms perform sexual reproduction, in which gametes from different sexes fuse to produce offspring. In most eukaryotes, one or both sex gametes are motile, and gametes actively approach each other to fuse. However, in flowering plants, the gametes of both sexes lack motility. Two sperm cells (male gametes) that are contained in a pollen grain are recessively delivered via pollen tube elongation. After the pollen tube bursts, sperm cells are released toward the egg and central cells (female gametes) within an ovule ( Fig. 1 ). The precise mechanism of sperm cell movement after the pollen tube bursts remains unknown. Ultimately, one sperm cell fuses with the egg cell and the other one fuses with the central cell, producing an embryo and an endosperm, respectively. Fertilization in which 2 sets of gamete fusion events occur, called double fertilization, has been known for over 100 y. The fact that each morphologically identical sperm cell precisely recognizes its fusion partner strongly suggests that an accurate gamete interaction system(s) exists in flowering plants.

Gamete attachment requires GEX2 for successful fertilization in Arabidopsis.

Fertilization requires recognition, attachment, and membrane fusion between gametes. In metazoans, rapidly evolving surface proteins contribute to gamete recognition and adhesion. Flowering plants evolved a double fertilization process wherein two immotile sperm cells are delivered to female gametes by the pollen tube, guided by elaborate communications between male and female reproductive organs. Once released, the sperm cells contact female gametes directly prior to gamete fusion. It remains unclear whether active gamete recognition and attachment mechanisms are required for double fertilization. Here, we provide functional characterization of Arabidopsis GAMETE EXPRESSED 2 (GEX2), which encodes a sperm-expressed protein of unknown function. GEX2 is localized to the sperm membrane and contains extracellular immunoglobulin-like domains, similar to gamete interaction factors in algae and mammals. Using a new in vivo assay, we demonstrate that GEX2 is required for gamete attachment, in the absence of which double fertilization is compromised. Ka/Ks analyses indicate relatively rapid evolution of GEX2, like other proteins involved in male and female interactions. We conclude that surface proteins involved in gamete attachment and recognition exist in plants with immotile gametes, similar to algae and metazoans. This conservation broadens the repertoire of research for plant reproduction factors to mechanisms demonstrated in animals.

Gamete membrane dynamics during double fertilization in Arabidopsis.

In the double fertilization of angiosperms, one sperm cell fertilizes an egg cell to produce a zygote, whereas the other sperm cell fertilizes a central cell to give rise to an endosperm. There is little information on gamete membrane dynamics during double fertilization even though the cell surface structure is critical for male and female gamete interactions. In a recent study, we analyzed gamete membrane behavior during double fertilization by live-cell imaging with Arabidopsis gamete membrane marker lines. We observed that the sperm membrane signals occasionally remained at the boundary of the female gametes after gamete fusion. In addition, sperm membrane signals entering the fertilized female gametes were detected. These findings suggested that plasma membrane fusion between male and female gametes occurred with the sperm internal membrane components entering the female gametes, and this was followed by plasmogamy.

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis.

Angiosperms have a unique sexual reproduction system called "double fertilization." One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.

Characterization of bacterial-type phosphoenolpyruvate carboxylase expressed in male gametophyte of higher plants.

BACKGROUND: Phosphoenolpyruvate carboxylase (PEPC) is a critical enzyme catalyzing the ß-carboxylation of phosphoenolpyruvate (PEP) to oxaloacetate, a tricarboxylic acid (TCA) cycle intermediate. PEPC typically exists as a Class-1 PEPC homotetramer composed of plant-type PEPC (PTPC) polypeptides, and two of the subunits were reported to be monoubiquitinated in germinating castor oil seeds. By the large-scale purification of ubiquitin (Ub)-related proteins from lily anther, two types of PEPCs, bacterial-type PEPC (BTPC) and plant-type PEPC (PTPC), were identified in our study as candidate Ub-related proteins. Until now, there has been no information about the properties of the PEPCs expressed in male reproductive tissues of higher plants. RESULTS: Expression analyses showed that lily BTPC (LlBTPC) and Arabidopsis BTPC (AtBTPC) were significantly expressed in pollen. The fusion protein AtBTPC-Venus localized in the cytoplasm of the vegetative cell (VC). Both LlBTPC and AtBTPC expression initiated after the last mitosis before pollen germination. Lily PTPC (LlPTPC) and monoubiquitinated LlPTPC (Ub-LlPTPC) remained at constant levels during pollen development. In late bicellular pollen of lily, LlBTPC forms a hetero-octameric Class-2 PEPC complex with LlPTPC to express PEPC activity. CONCLUSION: Our results suggest that an LlBTPC:Ub-LlPTPC:LlPTPC complex is formed in the VC cytoplasm during late pollen development. Both LlBTPC and AtBTPC expression patterns are similar to the patterns of the appearance of storage organelles during pollen development in lily and Arabidopsis, respectively. Therefore, BTPC is thought to accelerate the metabolic flow for the synthesis of storage substances during pollen maturation. Our study provides the first characterization of BTPC in pollen, the male gametophyte of higher plants.

Isolation and identification of ubiquitin-related proteins from Arabidopsis seedlings.

The majority of proteins in eukaryotic cells are modified according to highly regulated mechanisms to fulfill specific functions and to achieve localization, stability, and transport. Protein ubiquitination is one of the major post-translational modifications occurring in eukaryotic cells. To obtain the proteomic dataset related to the ubiquitin (Ub)-dependent regulatory system in Arabidopsis, affinity purification with an anti-Ub antibody under native condition was performed. Using MS/MS analysis, 196 distinct proteins represented by 251 distinct genes were identified. The identified proteins were involved in metabolism (23.0%), stress response (21.4%), translation (16.8%), transport (6.7%), cell morphology (3.6%), and signal transduction (1.5%), in addition to proteolysis (16.8%) to which proteasome subunits (14.3%) is included. On the basis of potential ubiquitination-targeting signal motifs, in-gel mobilities, and previous reports, 78 of the identified proteins were classified as ubiquitinated proteins and the rest were speculated to be associated proteins of ubiquitinated proteins. The degradation of three proteins predicted to be ubiquitinated proteins was inhibited by a proteasome inhibitor, suggesting that the proteins were regulated by Ub/proteasome-dependent proteolysis.

Identification of multiple highly similar XIP-type xylanase inhibitor genes in hexaploid wheat.

In hexaploid wheat, Xip-I is the only XIP-type xylanase inhibitor gene whose expression and function have been characterized in detail. Here we demonstrate the existence of new XIP-type genes with the identification of Xip-R1 and Xip-R2 in the root cDNAs. Southern blot analysis with the Xip-R1 probe revealed that XIP-type genes comprised a significantly greater gene family than previously speculated on in studies with the Xip-I probe. The transcript level of Xip-R genes was increased upon an inoculation with Erysiphe graminis in the leaves, but not with Fusarium graminearum in the spikelets. RT-PCR with the RNA samples followed by extensive sequencing of the cloned amplified products revealed the presence of 12 highly similar Xip-R genes. Among these genes, Xip-R1 was the only predominant Xip-R family member induced to express in response to E. graminis. XIP-R1 was located in the apoplastic space and inhibited family 11 xylanases, but the protein did not show chitinolytic activity. These results suggest that hexaploid wheat has a large family of XIPs in its genome, but that only some of them are expressed for plant defense in limited tissues.

Reduced contamination by the Fusarium mycotoxin zearalenone in maize kernels through genetic modification with a detoxification gene.

Maize is subject to ear rot caused by toxigenic Aspergillus and Fusarium species, resulting in contamination with aflatoxins, fumonisins, trichothecenes, and zearalenone (ZEN). The trichothecene group and ZEN mycotoxins are produced by the cereal pathogen Fusarium graminearum. A transgenic detoxification system for the elimination of ZEN was previously developed using an egfp::zhd101 gene (gfzhd101), encoding an enhanced green fluorescent protein fused to a ZEN-degrading enzyme. In this study, we produced a transgenic maize line expressing an intact copy of gfzhd101 and examined the feasibility of transgene-mediated detoxification in the kernels. ZEN-degrading activity has been detected in transgenic kernels during seed maturation (for a period of 6 weeks after pollination). The level of detoxification activity was unaltered after an additional storage period of 16 weeks at 6 degrees C. When the seeds were artificially contaminated by immersion in a ZEN solution for 48 h at 28 degrees C, the total amount of the mycotoxin in the transgenic seeds was uniformly reduced to less than 1/10 of that in the wild type. The ZEN in the transgenic maize kernels was also efficiently decontaminated under conditions of lower water activity (aw) and temperature; e.g., 16.9 microg of ZEN was removed per gram of seed within 48 h at an aw of 0.90 at 20 degrees C. F. graminearum infection assays demonstrated an absence of ZEN in the transgenic maize seeds, while the mycotoxin accumulated in wild-type kernels under the same conditions. Transgene-mediated detoxification may offer simple solutions to the problems of mycotoxin contamination in maize.