Synergid Cells Research Articles

Apomictic development during different flower development stages in Crataegus tanacetifolia (Lam.) Pers., endemic to Turkey

In this study, aposporic apomictic development and its relation to the different flower development stages were investigated by light and fluorescence microscopy in Crataegus tanacetifolia (Lam.) Pers. (Rosaceae). At pre-anthesis stage, aposporic initial cell differentiated at the somatic nucellus tissue shortly after the megaspore mother cell formation. The volume of aposporic initial cell increased during the generation of dyad and megaspore tetrad respectively by regular meiosis. At this stage, linear megaspore tetrad and vacuolated aposporic initial cell were located side by side into the same ovule. At anthesis stage, before pollination, four nucleated aposporic embryo sac was formed while sexual development came to end by atrophy of megaspores completely. At this stage, atrophied megaspores and two nucleated aposporic embryo sac were located side by side into the same ovule. At post-anthesis stage, pollination still had not begun and ovule contained only eight nucleated aposporic embryo sac. Mature aposporic embryo sac was composed of two synergid cells and one egg cell on the micropylar side, three antipodal cells on the chalazal side and a central cell with two polar nuclei in the middle of the sac. The absence of filiform apparatus in the synergid cells was quite remarkable. No callose accumulation around the aposporic initial cell was observed in any development stage. Pollination started shortly after the proembryo formation. Embryo and endosperm developed without fertilization due to the problems encountered in reaching pollen tubes to the ovary.

Fertilized egg cells secrete endopeptidases to avoid polytubey.

Upon gamete fusion, animal egg cells secrete proteases from cortical granules to establish a fertilization envelope as a block to polyspermy1-4. Fertilization in flowering plants is more complex and involves the delivery of two non-motile sperm cells by pollen tubes5,6. Simultaneous penetration of ovules by multiple pollen tubes (polytubey) is usually avoided, thus indirectly preventing polyspermy7,8. How plant egg cells regulate the rejection of extra tubes after successful fertilization is not known. Here we report that the aspartic endopeptidases ECS1 and ECS2 are secreted to the extracellular space from a cortical network located at the apical domain of the Arabidopsis egg cell. This reaction is triggered only after successful fertilization. ECS1 and ECS2 are exclusively expressed in the egg cell and transcripts are degraded immediately after gamete fusion. ECS1 and ESC2 specifically cleave the pollen tube attractor LURE1. As a consequence, polytubey is frequent in ecs1ecs2 double mutants. Ectopic secretion of these endopeptidases from synergid cells led to a decrease in the levels of LURE1 and reduced the rate of pollen tube attraction. Together, these findings demonstrate that plant egg cells sense successful fertilization and elucidate a mechanism as to how a relatively fast post-fertilization block to polytubey is established by fertilization-induced degradation of attraction factors.

Dynamics of the cell fate specifications during female gametophyte development in Arabidopsis.

The female gametophytes of angiosperms contain cells with distinct functions, such as those that enable reproduction via pollen tube attraction and fertilization. Although the female gametophyte undergoes unique developmental processes, such as several rounds of nuclear division without cell plate formation and final cellularization, it remains unknown when and how the cell fate is determined during development. Here, we visualized the living dynamics of female gametophyte development and performed transcriptome analysis of individual cell types to assess the cell fate specifications in Arabidopsis thaliana. We recorded time lapses of the nuclear dynamics and cell plate formation from the 1-nucleate stage to the 7-cell stage after cellularization using an in vitro ovule culture system. The movies showed that the nuclear division occurred along the micropylar-chalazal (distal-proximal) axis. During cellularization, the polar nuclei migrated while associating with the forming edge of the cell plate, and then, migrated toward each other to fuse linearly. We also tracked the gene expression dynamics and identified that the expression of MYB98pro::GFP-MYB98, a synergid-specific marker, was initiated just after cellularization in the synergid, egg, and central cells and was then restricted to the synergid cells. This indicated that cell fates are determined immediately after cellularization. Transcriptome analysis of the female gametophyte cells of the wild-type and myb98 mutant revealed that the myb98 synergid cells had egg cell-like gene expression profiles. Although in myb98, egg cell-specific gene expression was properly initiated in the egg cells only after cellularization, but subsequently expressed ectopically in one of the 2 synergid cells. These results, together with the various initiation timings of the egg cell-specific genes, suggest complex regulation of the individual gametophyte cells, such as cellularization-triggered fate initiation, MYB98-dependent fate maintenance, cell morphogenesis, and organelle positioning. Our system of live-cell imaging and cell type-specific gene expression analysis provides insights into the dynamics and mechanisms of cell fate specifications in the development of female gametophytes in plants.

Plant egg cell fate determination depends on its exact position in female gametophyte

Plant fertilization involves both an egg cell, which fuses with a sperm cell, and synergid cells, which guide pollen tubes for sperm cell delivery. Therefore, egg and synergid cell functional specifications are prerequisites for successful fertilization. However, how the egg and synergid cells, referred to as the "egg apparatus," derived from one mother cell develop into distinct cell types remains an unanswered question. In this report, we show that the final position of the nuclei in female gametophyte determines the cell fate of the egg apparatus. We established a live imaging system to visualize the dynamics of nuclear positioning and cell identity establishment in the female gametophyte. We observed that free nuclei should migrate to a specific position before egg apparatus specialization. Artificial changing in the nuclear position on disturbance of the actin cytoskeleton, either in vitro or in vivo, could reset the cell fate of the egg apparatus. We also found that nuclei of the same origin moved to different positions and then showed different cell identities, whereas nuclei of different origins moved to the same position showed the same cell identity, indicating that the final positions of the nuclei, rather than specific nucleus lineage, play critical roles in the egg apparatus specification. Furthermore, the active auxin level was higher in the egg cell than in synergid cells. Auxin transport inhibitor could decrease the auxin level in egg cells and impair egg cell identity, suggesting that directional and accurate auxin distribution likely acts as a positional cue for egg apparatus specialization.

Developmental, ultrastructural and cytochemical investigations of the female gametophyte in Sedum rupestre L. (Crassulaceae)

This article describes the development of female gametophyte in Sedum rupestre L. New embryological information about the processes of megasporogenesis and megagametogenesis provided in this paper expand the current knowledge about the embryology of the studied species. S. rupestre is characterized by monosporic megasporogenesis and the formation of Polygonum–type embryo sac. The process of megasporogenesis is initiated by one megaspore mother cell, resulting in the formation of a triad of cells after meiosis and cytokinesis. The functional megaspore, which is located chalazally, is a mononuclear cell present next to the megaspore in the centre of the triad. Only one of the two non-functional cells of the triad is binucleate, which occur at the micropylar pole. In this paper, we explain the functional ultrastructure of the female gametophytic cells in S. rupestre. Initially, the cytoplasm of the gametophytic cells does not differ from each other; however, during differentiation, the cells reveal different morphologies. The antipodals and the synergids gradually become organelle-rich and metabolically active. The antipodal cells participate in the absorption and transport of nutrients from the nucellar cells towards the megagametophyte. Their ultrastructure shows the presence of plasmodesmata with electron-dense material, which is characteristic of Crassulaceae, and wall ingrowths in the outer walls. The ultrastructure of synergid cells is characterized by the presence of filiform apparatus and cytoplasm with active dictyosomes, abundant profiles of endoplasmic reticulum and numerous vesicles, which agrees with their main function—the secretion of pollen tube attractants. Reported data can be used to resolve the current taxonomic problems within the genus Sedum ser. Rupestria.

Development of the female gametophyte of Agave colimana, subgenus Littaea (Asparagaceae)1

Agave colimana, subgenus Littaea (Asparagaceae) is a wild plant that belongs to the Asparagaceae family, and in this species, the formation of its female reproductive system is unknown, probably because it has no commercial use. However, A. colimana recently has been considered as a candidate for marketing as a succulent plant because of its floral and growth architecture. Knowledge about aspects of the formation of the embryo sac is useful for understanding its sexual reproduction, which is important for propagation by seed and breeding purposes. Megasporogenesis and megagametogenesis were studied using Feulgen staining under confocal microscopy. Megasporogenesis resulted in the formation of a linear tetrad with the functional megaspore attached to the chalazal end. Subsequently, megagametogenesis from the functional megaspore was followed by three mitotic divisions generating an embryo sac with seven cells: two synergid cells, one egg cell, three antipodal cells, and one central cell with a 2n nucleus. The embryo sac of Agave colimana was found to be monosporic of the Polygonum-type considered as the typical type in the majority of angiosperm plants. Also, abnormal embryo sacs were observed, following possible errors during meiosis.

Interaction Between AtCML9 and AtMLO10 Regulates Pollen Tube Development and Seed Setting.

In higher-plant reproduction, the compatibility of pollen tube germination in the pistil is essential for successful double fertilization. It has been reported that Mildew Locus O (MLO) family gene NTA (MLO7), expressing in synergid cells, can correctly guide pollen tubes. However, the molecular mechanism underlying the interacting partners to MLOs in the fertilization is still unknown. In our study, we identified the direct protein interaction between CML9 and MLO10 within a non-canonical CaMBD. In GUS reporter assays, CML9 expresses in a high level in pollens, whereas MLO10 can be specifically detected in stigma which reaches up to a peaking level before fertilization. Therefore, the spatio-temporal expression patterns of MLO10 and CML9 are required for the time-window of pollination. When we observed the pollen germination in vitro, two cml9 mutant alleles dramatically reduced germination rate by 15% compared to wild-type. Consistently, the elongation rate of pollen tubes in planta was obviously slow while manually pollinating cml9-1 pollens to mlo10-1 stigmas. Additionally, cml9-1 mlo10-1 double mutant alleles had relatively lower rate of seed setting. Taken together, protein interaction between MLO10 and CML9 is supposed to affect pollen tube elongation and further affect seed development.

Single-cell RNA-seq analysis reveals ploidy-dependent and cell-specific transcriptome changes in Arabidopsis female gametophytes

BackgroundPolyploidy provides new genetic material that facilitates evolutionary novelty, species adaptation, and crop domestication. Polyploidy often leads to an increase in cell or organism size, which may affect transcript abundance or transcriptome size, but the relationship between polyploidy and transcriptome changes remains poorly understood. Plant cells often undergo endoreduplication, confounding the polyploid effect.ResultsTo mitigate these effects, we select female gametic cells that are developmentally stable and void of endoreduplication. Using single-cell RNA sequencing (scRNA-seq) in Arabidopsis thaliana tetraploid lines and isogenic diploids, we show that transcriptome abundance doubles in the egg cell and increases approximately 1.6-fold in the central cell, consistent with cell size changes. In the central cell of tetraploid plants, DEMETER (DME) is upregulated, which can activate PRC2 family members FIS2 and MEA, and may suppress the expression of other genes. Upregulation of cell size regulators in tetraploids, including TOR and OSR2, may increase the size of reproductive cells. In diploids, the order of transcriptome abundance is central cell, synergid cell, and egg cell, consistent with their cell size variation. Remarkably, we uncover new sets of female gametophytic cell-specific transcripts with predicted biological roles; the most abundant transcripts encode families of cysteine-rich peptides, implying roles in cell-cell recognition during double fertilization.ConclusionsTranscriptome in single cells doubles in tetraploid plants compared to diploid, while the degree of change and relationship to the cell size depends on cell types. These scRNA-seq resources are free of cross-contamination and are uniquely valuable for advancing plant hybridization, reproductive biology, and polyploid genomics.

PRP8A and PRP8B spliceosome subunits act coordinately to control pollen tube attraction in Arabidopsis thaliana.

Precise guided pollen tube growth by the female gametophyte is a prerequisite for successful sexual reproduction in flowering plants. Cysteine-rich proteins (CRPs) secreted from the embryo sac are known pollen tube attractants perceived by pollen tube receptor-like kinases. How pre-mRNA splicing facilitates this cell-to-cell communication is not understood. Here, we report a novel function of Pre-mRNA PROCESSING factor 8 paralogs, PRP8A and PRP8B, as regulators of pollen tube attraction. Double mutant prp8a prp8b ovules cannot attract pollen tubes, and prp8a prp8b pollen tubes fail to sense the ovule's attraction signals. Only 3% of ovule-expressed genes were misregulated in prp8a prp8b Combination of RNA sequencing and the MYB98/LURE1.2-YFP reporter revealed that the expression of MYB98, LUREs and 49 other CRPs were downregulated, suggesting loss of synergid cell fate. Differential exon usage and intron retention analysis revealed autoregulation of PPR8A/PRP8B splicing. In vivo, PRP8A co-immunoprecipitates with splicing enhancer AtSF3A1, suggesting involvement of PRP8A in 3'-splice site selection. Our data hint that the PRP8A/PRP8B module exhibits spliceosome autoregulation to facilitate pollen tube attraction via transcriptional regulation of MYB98, CRPs and LURE pollen tube attractants.

Semi-In Vivo Assay for Pollen Tube Attraction.

In flowering plants, each pollen tube delivers two sperm cells into the ovule to complete double fertilization. During the process, pollen tubes need to be navigated into the ovule, where accurate and complex pre-ovule guidance and ovule guidance are required. In recent years, different methods have been established to study those genes involved in the regulation of pollen tube guidance. Semi-in vivo ovule targeting mimics in vivo pollen tube micropylar guidance, and the semi-in vivo ovule targeting assay has been used to investigate function of genes involved in micropylar guidance. Moreover, the ovule targeting assay is the best way to do live cell imaging, which facilitates observation of pollen tube reception, synergid cell degeneration, and semi-in vivo gamete fusion. Meanwhile, semi-in vivo pollen tube attraction assay is another useful method to directly determine whether a certain molecule has pollen tube attraction activity.

Cytoplasmic ribosomal protein L14B is essential for fertilization in Arabidopsis

Plant cytoplasmic ribosomal proteins not only participate in protein synthesis, but also have specific roles in developmental regulation. However, the high heterogeneity of plant ribosome makes our understanding of these proteins very limited. Here we reported that RPL14B, a component of the ribosome large subunit, is critical for fertilization in Arabidopsis. RPL14B is existed in a majority of organs and tissues. No homozygous rpl14b mutant is available, indicating that RPL14B is irreplaceable for sexual reproduction. Smaller–sized rpl14b pollens could germinate normally, but pollen tube competitiveness is grievously weakened. Beside, cell fate specification is impaired in female gametophytes from heterozygous rpl14b/RPL14B ovules, resulting in defect of micropylar pollen tube attraction. However, this defect could be restored by restricted expression of RPL14B in synergid cells. Successful fertilization requires normal pollen tube growth and precise pollen tube guidance. Thus our results show a novel role of RPL14B in fertilization and shed new light on regulatory mechanism of pollen tube growth and precise pollen tube guidance.

CrRLK1L receptor-like kinases HERK1 and ANJEA are female determinants of pollen tube reception.

Communication between the gametophytes is vital for angiosperm fertilisation. Multiple CrRLK1L-type receptor kinases prevent premature pollen tube burst, while another CrRLK1L protein, FERONIA (FER), is required for pollen tube reception in the female gametophyte. We report here the identification of two additional CrRLK1L homologues, HERCULES RECEPTOR KINASE 1 (HERK1) and ANJEA (ANJ), which act redundantly to promote pollen tube growth arrest at the synergid cells. HERK1 and ANJ localise to the filiform apparatus of the synergid cells in unfertilised ovules, and in herk1 anj mutants, a majority of ovules remain unfertilised due to pollen tube overgrowth, together indicating that HERK1 and ANJ act as female determinants for fertilisation. As in fer mutants, the synergid cell-specific, endomembrane protein NORTIA (NTA) is not relocalised after pollen tube reception; however, unlike fer mutants, reactive oxygen species levels are unaffected in herk1 anj double mutants. Both ANJ and HERK1 associate with FER and its proposed co-receptor LORELEI (LRE) in planta. Together, our data indicate that HERK1 and ANJ act with FER to mediate female-male gametophyte interactions during plant fertilisation.

REM34 and REM35 Control Female and Male Gametophyte Development in Arabidopsis thaliana.

The REproductive Meristem (REM) gene family encodes for transcription factors belonging to the B3 DNA binding domain superfamily. In Arabidopsis thaliana, the REM gene family is composed of 45 members, preferentially expressed during flower, ovule, and seed developments. Only a few members of this family have been functionally characterized: VERNALIZATION1 (VRN1) and, most recently, TARGET OF FLC AND SVP1 (TFS1) regulate flowering time and VERDANDI (VDD), together with VALKYRIE (VAL) that control the death of the receptive synergid cell in the female gametophyte. We investigated the role of REM34, REM35, and REM36, three closely related and linked genes similarly expressed in both female and male gametophytes. Simultaneous silencing by RNA interference (RNAi) caused about 50% of the ovules to remain unfertilized. Careful evaluation of both ovule and pollen developments showed that this partial sterility of the transgenic RNAi lines was due to a postmeiotic block in both female and male gametophytes. Furthermore, protein interaction assays revealed that REM34 and REM35 interact, which suggests that they work together during the first stages of gametogenesis.

Development of a Heat-Inducible Gene Expression System Using Female Gametophytes of Arabidopsis thaliana.

Female gametophyte (FG) is crucial for reproduction in flowering plants. Arabidopsis thaliana produces Polygonum-type FGs, which consist of an egg cell, two synergid cells, three antipodal cells and a central cell. Egg cell and central cell are the two female gametes that give rise to the embryo and surrounding endosperm, respectively, after fertilization. During the development of a FG, a single megaspore produced by meiosis undergoes three rounds of mitosis to produce an eight-nucleate cell. A seven-celled FG is formed after cellularization. The central cell initially contains two polar nuclei that fuse during female gametogenesis to form the secondary nucleus. In this study, we developed a gene induction system for analyzing the functions of various genes in developing Arabidopsis FGs. This system allows transgene expression in developing FGs using the heat-inducible Cre-loxP recombination system and FG-specific embryo sac 2 (ES2) promoter. Efficient gene induction was achieved in FGs by incubating flower buds and isolated pistils at 35�C for short periods of time (1-5 min). Gene induction was also induced in developing FGs by heat treatment of isolated ovules using the infrared laser-evoked gene operator (IR-LEGO) system. Expression of a dominant-negative mutant of Sad1/UNC84 (SUN) proteins in developing FGs using the gene induction system developed in this study caused defects in polar nuclear fusion, indicating the roles of SUN proteins in this process. This strategy represents a new tool for analyzing the functions of genes in FG development and FG functions.

Gene expression and localization of arabinogalactan proteins during the development of anther, ovule, and embryo in rice.

Arabinogalactan proteins (AGPs) are hyperglycosylated members of the hydroxyproline-rich glycoprotein (HRGP) superfamily and are widely distributed throughout the plant kingdom. In Oryza sativa (rice), the gene expression and biological function of AGPs only have received minimal research attention. Here, we used qRT-PCR to detect the expression patterns of OsAGP genes in various organs, and found that six genes were preferentially expressed in panicles, three genes were specifically expressed in anthers, and one gene in the stigma. Furthermore, using four specific monoclonal antibodies (JIM8, JIM13, LM2, MAC207), we observed the distribution of AGPs in rice anthers, ovules, and embryos. In anthers, the strong fluorescence signals of AGPs were present in tapetum cells, pollen mother cells, and mature pollens, suggesting that AGPs might be related to the development of anther and pollen. In ovules, signals of AGPs were specifically distributed in the three micropylar megaspores of the tetrad, and with intense signals in the egg cell and synergid cells in the mature embryo sac. This suggests that AGPs may be involved in megaspore determination and double fertilization. In embryos, the immunological signals of AGPs appeared in peripheral and inner cells at the early stage, and in the scutellum, plumule, and radicle at the late stage, indicating that AGPs may be associated with organ differentiation and maturation of embryos. In this study, we revealed that AGPs were widely distributed in rice anthers, ovules, and embryos, which lays a foundation for the functional investigation of AGPs in various processes of sexual reproduction.

Reproductive potential of the functionally female native Croatian grapevine 'Grk bijeli'

A native Croatian grapevine 'Grk bijeli', sharing a parent-offspring relationships with 'Tribidrag' (aka 'Zinfandel'), is grown exclusively on the Adriatic island of Korcula. It is one of the grape cultivars with female-only functional flowers, causing reduced fertilisation and leads to problems in grape production and wine quality. A typical cluster of 'Grk bijeli' at maturity contains fully developed seeded berries, in addition to a highly variable proportion of undersized seedless berries. The aim of this study was to identify the cause of the reduced reproduction potential of 'Grk bijeli' in order to establish a better growing environment for improved yield and grape quality. 'Grk bijeli' female gametophyte develops normaly and at maturity contains both egg and central cell, together with two synergid cells and three antipodal cells. On the other hand, 'Grk bijeli' pollen grains show developmental deviations. Specifically, most of the pollen grains undergo the first pollen mitosis and contain one vegetative cell and one generative cell, while 20 % of ungerminating grains also accomplish the second pollen mitosis, giving rise to two sperm cells and one vegetative cell. Morover, 'Grk bijeli' pollen has acolporate morphology, which prevents germination and contributes to reduced reproduction. Furthermore, fertilisation after pollination with other varieties results in various degrees of ovule abortion depending on the pollinator, revealing Croatian cultivars 'Plavac mali crni' and 'Posip bijeli' as favourable varieties. Although this study describes a highly valuable cultivar of local importance, it also contributes to fundamental knowledge of grapevine reproductive biology and offers a strategy for improvement of wine production and oenological performance of semi-fertile varieties in general.

Friend or foe: Signaling mechanisms during double fertilization in flowering seed plants.

Since the first description of double fertilization 120 years ago, the processes of pollen tube growth and guidance, sperm cell release inside the receptive synergid cell, as well as fusion of two sperm cells to the female gametes (egg and central cell) have been well documented in many flowering plants. Especially microscopic techniques, including live cell imaging, were used to visualize these processes. Molecular as well as genetic methods were applied to identify key players involved. However, compared to the first 11 decades since its discovery, the past decade has seen a tremendous advancement in our understanding of the molecular mechanisms regulating angiosperm fertilization. Whole signaling networks were elucidated including secreted ligands, corresponding receptors, intracellular interaction partners, and further downstream signaling events involved in the cross-talk between pollen tubes and their cargo with female reproductive cells. Biochemical and structural biological approaches are now increasingly contributing to our understanding of the different signaling processes required to distinguish between compatible and incompatible interaction partners. Here, we review the current knowledge about signaling mechanisms during above processes with a focus on the model plants Arabidopsis thaliana and Zea mays (maize). The analogy that many of the identified "reproductive signaling mechanisms" also act partly or fully in defense responses and/or cell death is also discussed.

Ethylene signaling is critical for synergid cell functional specification and pollen tube attraction

ETHYLENE INSENSITIVE 3 (EIN3) is a key regulator of ethylene signaling, and EIN3-BINDING F-BOX1 (EBF1) and EBF2 are responsible for EIN3 degradation. Previous reports have shown that the ebf1 ebf2 double homozygous mutant cannot be identified. In this study, the genetic analysis revealed that the ebf1 ebf2 female gametophyte is defective. The pollination experiment showed that ebf1 ebf2 ovules failed to attract pollen tubes. In female gametophyte/ovule, the synergid cell is responsible for pollen tube attraction. Observation of the pEIN3::EIN3-GFP transgenic lines showed that EIN3 signal was over-accumulated at the micropylar end of ebf1 ebf2 female gametophyte. The overexpression of stabilized EIN3 in synergid cell led to the defect of pollen tube guidance. These results suggested that the over-accumulated EIN3 in ebf1 ebf2 synergid cell blocks its pollen tube attraction which leads to the failure of ebf1 ebf2 homozygous plant. We identified that EIN3 directly activated the expression of a sugar transporter, SENESCENCE-ASSOCIATED GENE29 (SAG29/SWEET15). Overexpression of SAG29 in synergid cells blocked pollen tube attraction, suggesting that SAG29 might play a role in ethylene signaling to repel pollen tube entry. Taken together, our study reveals that strict control of ethylene signaling is critical for the synergid cell function during plant reproduction.

Studies on development of megasporogenesis and female gametophyte of Bambusa multiplex.

In order to discuss whether the development of the female organ is normal, the development of megasporogenesis and female gametophyte of Bambusa multiplex was studied by the method of traditional paraffin section and SEM. The results showed that the ovary had one anatropous ovule, bitegmic and tenuinucellate. A female archesporial cell developed into megaspore mother cell directly, linear tetrad. The functional megaspore near the micropyle developed into mature embryo sac with eight nucleuses in 7 cells, including 3 antipodal cells arranged transversely near the chalazal and egg apparatus (1 egg cell and 2 synergid cells) near the micropylar end and a central cell (2 polar nucleus). In addition, the corresponding development relationship between pistil and stamen was established, which discovered the anthers and stigmas of B. multiplex matured at the same time. The corresponding development relationship between pistil and stamen may provide the reference for the developmental phase of embryo sac according to the anther length. During the pistil development process abnormal structures of ovaries may be one of the reasons of the low seed rate.

Protoplast-Esculin Assay as a New Method to Assay Plant Sucrose Transporters: Characterization of AtSUC6 and AtSUC7 Sucrose Uptake Activity in Arabidopsis Col-0 Ecotype.

The best characterized function of sucrose transporters of the SUC family in plants is the uptake of sucrose into the phloem for long-distance transport of photoassimilates. This important step is usually performed by one specific SUC in every species. However, plants possess small families of several different SUCs which are less well understood. Here, we report on the characterization of AtSUC6 and AtSUC7, two members of the SUC family in Arabidopsis thaliana. Heterologous expression in yeast (Saccharomyces cerevisiae) revealed that AtSUC6Col-0 is a high-affinity H+-symporter that mediates the uptake of sucrose and maltose across the plasma membrane at exceptionally low pH values. Reporter gene analyses revealed a strong expression of AtSUC6Col-0 in reproductive tissues, where the protein product might contribute to sugar uptake into pollen tubes and synergid cells. A knockout of AtSUC6 did not interfere with vegetative development or reproduction, which points toward physiological redundancy of AtSUC6Col-0 with other sugar transporters. Reporter gene analyses showed that AtSUC7Col-0 is expressed in roots and pollen tubes and that this sink specific expression of AtSUC7Col-0 is regulated by intragenic regions. Transport activity of AtSUC7Col-0 could not be analyzed in baker’s yeast or Xenopus oocytes because the protein was not correctly targeted to the plasma membrane in both heterologous expression systems. Therefore, a novel approach to analyze sucrose transporters in planta was developed. Plasma membrane localized SUCs including AtSUC6Col-0 and also sucrose specific SWEETs were able to mediate transport of the fluorescent sucrose analog esculin in transformed mesophyll protoplasts. In contrast, AtSUC7Col-0 is not able to mediate esculin transport across the plasma membrane which implicates that AtSUC7Col-0 might be a non-functional pseudogene. The novel protoplast assay provides a useful tool for the quick and quantitative analysis of sucrose transporters in an in planta expression system.