RALF peptide signaling controls the polytubey block in Arabidopsis.

Fertilization of an egg by multiple sperm (polyspermy) leads to lethal genome imbalance and chromosome segregation defects. In Arabidopsis thaliana, the block to polyspermy is facilitated by a mechanism that prevents polytubey (the arrival of multiple pollen tubes to one ovule). We show here that FERONIA, ANJEA, and HERCULES RECEPTOR KINASE 1 receptor-like kinases located at the septum interact with pollen tube-specific RALF6, 7, 16, 36, and 37 peptide ligands to establish this polytubey block. The same combination of RALF (rapid alkalinization factor) peptides and receptor complexes controls pollen tube reception and rupture inside the targeted ovule. Pollen tube rupture releases the polytubey block at the septum, which allows the emergence of secondary pollen tubes upon fertilization failure. Thus, orchestrated steps in the fertilization process in Arabidopsis are coordinated by the same signaling components to guarantee and optimize reproductive success.

Glucuronidation of type II arabinogalactan polysaccharides function in sexual reproduction of Arabidopsis.

Arabinogalactan proteins (AGPs) are complex, hyperglycosylated plant cell wall proteins with little known about the biological roles of their glycan moieties in sexual reproduction. Here, we report that GLCAT14A, GLCAT14B, and GLCAT14C, three enzymes responsible for the addition of glucuronic acid residues to AGPs, function in pollen development, polytubey block, and normal embryo development in Arabidopsis. Using biochemical and immunolabeling techniques, we demonstrated that the loss of function of the GLCAT14A, GLCAT14B, and GLCAT14C genes resulted in disorganization of the reticulate structure of the exine wall, abnormal development of the intine layer, and collapse of pollen grains in glcat14a/b and glcat14a/b/c mutants. Synchronous development between locules within the same anther was also lost in some glcat14a/b/c stamens. In addition, we observed excessive attraction of pollen tubes targeting glcat14a/b/c ovules, indicating that the polytubey block mechanism was compromised. Monosaccharide composition analysis revealed significant reductions in all sugars in glcat14a/b and glcat14a/b/c mutants except for arabinose and galactose, while immunolabeling showed decreased amounts of AGP sugar epitopes recognized by glcat14a/b and glcat14a/b/c mutants compared with the wild type. This work demonstrates the important roles that AG glucuronidation plays in Arabidopsis sexual reproduction and reproductive development.

OsMTD2-mediated reactive oxygen species (ROS) balance is essential for intact pollen-tube elongation in rice.

The highly specialized haploid male gametophyte-pollen consist of two sperm cells and a large vegetative cell. Successful fertilization requires proper growth timing and rupture of the pollen tube until it delivers sperm cells, which occur immediately after a pollen grain hydrates. Although a tight regulation on polar cell-wall expansion of the pollen tube is fundamentally important, the underlying molecular mechanism remains largely unknown, especially in crop plants. Here, we characterized the function of male-gene transfer defective 2 (OsMTD2) gene in rice (Oryza sativa), which belongs to the plant-specific receptor-like kinase, the CrRLK1L family. We demonstrated that OsMTD2 is an essential male factor participating in pollen-tube elongation based on genetic evidence and physiological observations. Because of unavailability of homozygous mutant via conventional methods, we used CRISPR-Cas9 system to obtain homozygous knockout mutant of OsMTD2. We were able to identify phenotypic changes including male sterility due to early pollen-tube rupture in the mutant. We observed that the production of reactive oxygen species (ROS) was dramatically reduced in mutants of OsMTD2 pollen grain and tubes with defective pectin distribution. Transcriptome analysis of osmtd2-2 versus wild-type anthers revealed that genes involved in defense responses, metabolic alteration, transcriptional and protein modification were highly upregulated in the osmtd2-2 mutant. Through yeast-two-hybrid screening, we found that OsMTD2 kinase interacts with E3 ligase SPL11. Taken together, we propose that OsMTD2 has crucial functions in promoting pollen-tube elongation through cell-wall modification, possibly by modulating ROS homeostasis during pollen-tube growth.

Turgor regulation defect 1 proteins play a conserved role in pollen tube reproductive innovation of the angiosperms.

Sexual reproduction in angiosperms is siphonogamous, and the interaction between pollen tube and pistil is critical for successful fertilization. Our previous study demonstrated that mutation of the Arabidopsis turgor regulation defect 1 (TOD1) gene leads to reduced male fertility, a result of retarded pollen tube growth in the pistil. TOD1 encodes a Golgi-localized alkaline ceramidase, a key enzyme for the production of sphingosine-1-phosphate (S1P), which is involved in the regulation of turgor pressure in plant cells. However, whether TOD1s play a conserved role in the innovation of siphonogamy is largely unknown. In this study, we provide evidence that OsTOD1, which is similar to AtTOD1, is also preferentially expressed in rice pollen grains and pollen tubes. OsTOD1 knockout results in reduced pollen tube growth potential in rice pistil. Both the OsTOD1 genomic sequence with its own promoter and the coding sequence under the AtTOD1 promoter can partially rescue the attod1 mutant phenotype. Furthermore, TOD1s from other angiosperm species can partially rescue the attod1 mutant phenotype, while TOD1s from gymnosperm species are not able to complement the attod1 mutant phenotype. Our data suggest that TOD1 acts conservatively in angiosperms, and this opens up an opportunity to dissect the role of sphingolipids in pollen tube growth in angiosperms.

AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube guidance.

Reproductive isolation is a prerequisite to form and maintain a new species. Multiple prezygotic and postzygotic reproductive isolation barriers have been reported in plants. In the model plant, Arabidopsis thaliana conspecific pollen tube precedence controlled by AtLURE1/PRK6-mediated signaling has been recently reported as a major prezygotic reproductive isolation barrier. By accelerating emergence of own pollen tubes from the transmitting tract, A. thaliana ovules promote self-fertilization and thus prevent fertilization by a different species. Taking advantage of a septuple atlure1null mutant, we now report on the role of AtLURE1/PRK6-mediated signaling for micropylar pollen tube guidance. Compared with wild-type (WT) ovules, atlure1null ovules displayed remarkably reduced micropylar pollen tube attraction efficiencies in modified semi-in vivo A. thaliana ovule targeting assays. However, when prk6 mutant pollen tubes were applied, atlure1null ovules showed micropylar attraction efficiencies comparable to that of WT ovules. These findings indicate that AtLURE1/PRK6-mediated signaling regulates micropylar pollen tube attraction in addition to promoting emergence of own pollen tubes from the transmitting tract. Moreover, semi-in vivo ovule targeting competition assays with the same amount of pollen grains from both A. thaliana and Arabidopsis lyrata showed that A. thaliana WT and xiuqiu mutant ovules are mainly targeted by own pollen tubes and that atlure1null mutant ovules are also entered to a large extent by A. lyrata pollen tubes. Taken together, we report that AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube attraction representing an additional prezygotic isolation barrier.

Treatment with spermidine alleviates the effects of concomitantly applied cold stress by modulating Ca2+, pH and ROS homeostasis, actin filament organization and cell wall deposition in pollen tubes of Camellia sinensis.

The aim of the current study was to examine the effect of spermidine treatment concomitant with cold stress on the elongation of Camellia sinensis pollen tube. When exogenous spermidine (0.05 mM) was applied concomitantly with cold stress, pollen germination rate and pollen tube length were significantly increased in comparison with cold stressed pollen tubes. In addition, spermidine treatment concomitantly with cold stress reduced pollen tube abnormalities induced by cold stress. Besides, cold-induced disorganizations of actin filaments were ameliorated after spermidine treatment along with cold stress because anisotropy levels of actin filaments in shank and apex of pollen tubes decreased. Changes in cold-induced callose distribution in the pollen tube cell wall were partially recovered after spermidine/cold stress treatment. Other cold-induced effects (decrease in Ca2+ content, reduction of pH gradient, accumulation of ROS) were reverted to adequate levels after spermidine treatment in conjunction with cold stress, indicating that pollen tubes are able to cope with stress. Thus, spermidine treatment reorganized the growth pattern of pollen tubes by modulating Ca2+ and ROS homeostasis, actin cytoskeleton organization, and cell wall deposition in Camellia sinensis pollen tubes under cold stress.

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.

Deficiency of very long chain alkanes biosynthesis causes humidity-sensitive male sterility via affecting pollen adhesion and hydration in rice.

Pollen adhesion and hydration are the earliest events of the pollen-stigma interactions, which allow compatible pollen to fertilize egg cells, but the underlying mechanisms are still poorly understood. Rice pollen are wind dispersed, and its pollen coat contains less abundant lipids than that of insect-pollinated plants. Here, we characterized the role of OsGL1-4, a rice member of the Glossy family, in pollen adhesion and hydration. OsGL1-4 is preferentially expressed in pollen and tapetal cells and is required for the synthesis of very long chain alkanes. osgl1-4 mutant generated apparently normal pollen but displayed excessively fast dehydration at anthesis and defective adhesion and hydration under normal condition, but the defective adhesion and hydration were rescued by high humidity. Gas chromatography-mass spectrometry analysis suggested that the humidity-sensitive male sterility of osgl1-4 was probably due to a significant reduction in C25 and C27 alkanes. These results indicate that very long chain alkanes are components of rice pollen coat and control male fertility via affecting pollen adhesion and hydration in response to environmental humidity. Moreover, we proposed that a critical point of water content in mature pollen is required for the initiation of pollen adhesion.

Arabinogalactan proteins mediate intercellular crosstalk in the ovule of apple flowers.

KEY MESSAGE: AGP-rich glycoproteins mediate pollen-ovule interactions and cell patterning in the embryo sac of apple before and after fertilization. Glycoproteins are significant players in the dialog that takes place between growing pollen tubes and the stigma and style in the angiosperms. Yet, information is scarce on their possible involvement in the ovule, a sporophytic organ that hosts the female gametophyte. Apple flowers have a prolonged lapse of time between pollination and fertilization, offering a great system to study the developmental basis of glycoprotein secretion and their putative role during the last stages of the progamic phase and early seed initiation. For this purpose, the sequential pollen tube elongation within the ovary was examined in relation to changes in arabinogalactan proteins (AGPs) in the tissues of the ovule before and after fertilization. To evaluate what of these changes are developmentally regulated, unpollinated and pollinated flowers were compared. AGPs paved the pollen tube pathway in the ovules along the micropylar canal, and the nucellus entrance toward the synergids, which also developmentally accumulated AGPs at the filiform apparatus. Glycoproteins vanished from all these tissues following pollen tube passage, strongly suggesting a role in pollen-ovule interaction. In addition, AGPs marked the primary cell walls of the haploid cells of the female gametophyte, and they further built up in the cell walls of the embryo sac and developing embryo, layering the interactive walls of the three generations hosted in the ovule, the maternal sporophytic tissues, the female gametophyte, and the developing embryo.

Identification of a role for an E6-like 1 gene in early pollen-stigma interactions in Arabidopsis thaliana.

KEY MESSAGE: We describe a function for a novel Arabidopsis gene, E6-like 1 (E6L1), that was identified as a highly expressed gene in the stigma and plays a role in early post-pollination stages. In Arabidopsis, successful pollen-stigma interactions are dependent on rapid recognition of compatible pollen by the stigmatic papillae located on the surface of the pistil and the subsequent regulation of pollen hydration and germination, and followed by the growth of pollen tubes through the stigma surface. Here we have described the function of a novel gene, E6-like 1 (E6L1), that was identified through the analysis of transcriptome datasets, as one of highest expressed genes in the stigma, and furthermore, its expression was largely restricted to the stigma and trichomes. The first E6 gene was initially identified as a highly expressed gene during cotton fiber development, and related E6-like predicted proteins are found throughout the Angiosperms. To date, no orthologous genes have been assigned a biological function. Both the Arabidopsis E6L1 and cotton E6 proteins are predicted to be secreted, and this was confirmed using an E6L1:RFP fusion construct. To further investigate E6L1's function, one T-DNA and two independent CRISPR-generated mutants were analyzed for compatible pollen-stigma interactions, and pollen hydration, pollen adhesion, and seed set were mildly impaired for the e6l1 mutants. This work identifies E6L1 as a novel stigmatic factor that plays a role during the early post-pollination stages in Arabidopsis.

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress.

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H2 DCFDA-staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into 'low-ROS' and 'high-ROS' subpopulations. Pollen germination assays following fluorescence-activated cell sorting revealed that the high-ROS pollen germinated with a frequency that was 35-fold higher than the low-ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose-dependent alterations in DCF-fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry-based approaches to investigate metabolic changes during stress responses in pollen.

Integrated metabolite profiling and transcriptome analysis reveals a dynamic metabolic exchange between pollen tubes and the style during fertilization of Brassica napus.

KEY MESSAGE: In this study, we analyzed the transcriptome and metabolite profile of the style to explore the essential metabolites and specific genes for pollen tube growth of B. napus in vivo. For sexual reproduction of flowering plants, pollen must germinate on the stigma and the pollen tube must grow through the style to deliver the sperm nuclei to the female gametophyte cells. During this process, the rapidly growing pollen tube can cover substantial distances. Despite the clear requirements for energy and cellular building blocks in this process, few studies have examined the role of metabolism in the style for pollen tube elongation. In this study, we comprehensively analyzed the transcriptome and metabolite profiles during pollen germination and pollen tube growth in the style in Brassica napus. We profiled the transcripts and metabolites stored in pollen and identified many transcripts related to metabolic pathways. Mature pollen contained low levels of nutrients, whereas the styles contained high levels of diverse nutrients. The levels of most nutrients in the style, especially metabolites for cell wall synthesis and energy metabolism, rapidly decreased at 2 h after pollination, along with pollen germination and pollen tube elongation through the style. A subset of genes involved in cell wall synthesis and nutrient transport were expressed specifically in styles at 1 h after pollination. These results demonstrated that successful fertilization involves the transcripts and nutrients stored in mature pollen, and specific gene expression and stored nutrients in the style. Therefore, these findings enhance our understanding of fertilization in B. napus.

Arabidopsis pollen tube germination and growth depend on the mitochondrial calcium uniporter complex.

The mitochondrial calcium uniporter complex (MCUc) was recently characterized in details in metazoans and consists of pore-forming units (MCUs) and regulatory factors that channel calcium (Ca2+ ) ion into the mitochondria. MCUs participate in many stress and developmentally related processes involving Ca2+ . Although multiple homologues of MCUs and one regulatory subunit are usually present in plants, the first functional characterization and contribution to Ca2+ related processes of these proteins have been reported recently. Here, we focused on two predicted Arabidopsis MCUs and studied their role in the germination and the growth of pollen tube, a tip-growing cell type highly dependent on Ca2+ homeostasis. Heterologous expression of MCU1 or MCU2 in yeast is sufficient to generate a mitochondrial Ca2+ influx. MCU1 and MCU2 fluorescent reporters are co-expressed in the vegetative cell mitochondria of the pollen grain but are undetectable in the embryo sac. We demonstrate that MCU1 and MCU2 can form a heterotypic complex. Phenotypic analyses revealed an impaired pollen tube germination and growth in vitro only for the mcu2 mutants suggesting a predominant role of MCU2. Our results show that mitochondrial Ca2+ controlled by MCUs is an additional player in Arabidopsis pollen tube germination and growth.

Effect of flower orientation on the male and female traits of Myrtillocactus geometrizans (Cactaceae).

Intra-individual variation in the production and size of reproductive traits has been documented in columnar cacti, being higher in equator-facing flowers. Such variation is attributed to the high amount of PAR intercepted by stems oriented towards the equator. Most studies focused on this phenomenon have documented the existence of intra-individual variation on traits associated with the female function; however, its impact on traits associated with the male function has been neglected. We tested the hypothesis that equator-facing flowers of Myrtillocactus geometrizans exhibit higher values on traits associated with both male and female functions than flowers facing against it. Number and size of anthers and ovaries, pollen:ovule ratio and number and quality of pollen grains (diameter, germinability, viability and pollen tube length) were estimated from reproductive structures facing north and south, and compared with t-tests between orientations. Number of anthers per flower, number of pollen grains per anther and per floral bud; pollen size, viability and germinability; pollen tube length; ovary length and pollen:ovule ratio were significantly higher in reproductive structures oriented towards the south (i.e. equator). These findings suggest that intra-individual variation in floral traits of M. geometrizans might be associated with different availability of resources in branches with contrasting orientation. Our results provide new evidence of the existence of a response to an orientation-dependent extrinsic gradient. To our knowledge, this is the first study documenting the existence of intra-individual variation on pollen quality and P:O ratio in Cactaceae species.

Temperatures during flower bud development affect pollen germination, self-incompatibility reaction and early fruit development of clementine (Citrus clementina Hort. ex Tan.).

One of the key environmental factors affecting plant reproductive systems is temperature. Characterising such effects is especially relevant for some commercially important genera such as Citrus. In this genus, failure of fertilisation results in parthenocarpic fruit development and seedlessness, which is a much-prized character. Here, we characterise the effects of temperature on flower and ovary development, and on pollen-pistil interactions in 'Comune' clementine (Citrus clementina Hort. ex Tan.). We examine flower bud development, in vitro pollen germination and pollen-pistil interaction at different temperatures (15, 20, 25 or 30 °C). These temperatures span the range from 'cold' to 'hot' weather during the flowering season in many citrus-growing regions. Temperature had a strong effect on flower and ovary development, pollen germination, and pollen tube growth kinetics. In particular, parthenocarpic fruit development (indicated by juice vesicle growth) was initiated early if flowers were exposed to warmer temperatures during anthesis. Exposure to different temperatures during flower bud development also alters expression of the self-incompatibility reaction. This affects the point in the pistil at which pollen tube growth is arrested and confirms the role of sub- and supra-optimal temperatures in determining the numbers of pollen tubes reaching the ovary.

Organizational Innovation of Apical Actin Filaments Drives Rapid Pollen Tube Growth and Turning.

Polarized tip growth is a fundamental cellular process in many eukaryotes. In this study, we examined the dynamic restructuring of the actin cytoskeleton and its relationship to vesicle transport during pollen tip growth in Arabidopsis. We found that actin filaments originating from the apical membrane form a specialized structure consisting of longitudinally aligned actin bundles at the cortex and inner cytoplasmic filaments with a distinct distribution. Using actin-based pharmacological treatments and genetic mutants in combination with FRAP (fluorescence recovery after photobleaching) technology to visualize the transport of vesicles within the growth domain of pollen tubes, we demonstrated that cortical actin filaments facilitate tip-ward vesicle transport. We also discovered that the inner apical actin filaments prevent backward movement of vesicles, thus ensuring that sufficient vesicles accumulate at the pollen tube tip to support the rapid growth of the pollen tube. The combinatorial effect of cortical and internal apical actin filaments perfectly explains the generation of the inverted "V" cone-shaped vesicle distribution pattern at the pollen tube tip. When pollen tubes turn, apical actin filaments at the facing side undergo depolymerization and repolymerization to reorient the apical actin structure toward the new growth direction. This actin restructuring precedes vesicle accumulation and changes in tube morphology. Thus, our study provides new insights into the functional relationship between actin dynamics and vesicle transport during rapid and directional pollen tube growth.

Arabidopsis aspartic proteases A36 and A39 play roles in plant reproduction.

Aspartic proteases (Aps, EC3.4.23) are one of the 4 major mechanistic classes of proteolytic enzymes with the conserved motifs Asp-Thr/Ser-Gly (DT/SG) at the active site and are activated at acidic pH. In Arabidopsis, 69 genes were identified as coding putative aspartic proteinases. However, little is known about most of these enzymes. Recently, we characterized 2 novel Arabidopsis Aps genes, A36 and A39, which encode 2 putative GPI-anchored pollen-high-expressed Aps. a36 a39 mutants display significant abortion. The pollen grains underwent apoptosis-like programmed cell death and the degeneration of female gametes was also appeared in the a36 a39 mutant. Besides, the pollen tube of a36 a39 has compromised micropylar guidance. A36 and A39 were membrane-anchored protein and co-localized with a reported GPI-anchored protein COBRA-LIKE 10 (COBL10). In apical region of a36 a39 pollen tubes cell wall, the abundance of highly methlyestered homogalacturonans and xyloglucans were significantly increased. These results indicated that A36 and A39 are vital factors involved in gametogenesis and pollen guidance in Arabidopsis.

The Tension-sensitive Ion Transport Activity of MSL8 is Critical for its Function in Pollen Hydration and Germination.

All cells respond to osmotic challenges, including those imposed during normal growth and development. Mechanosensitive (MS) ion channels provide a conserved mechanism for regulating osmotic forces by conducting ions in response to increased membrane tension. We previously demonstrated that the MS ion channel MscS-Like 8 (MSL8) is required for pollen to survive multiple osmotic challenges that occur during the normal process of fertilization, and that it can inhibit pollen germination. However, it remained unclear whether these physiological functions required ion flux through a mechanically gated channel provided by MSL8. We introduced two point mutations into the predicted pore-lining domain of MSL8 that disrupted normal channel function in different ways. The Ile711Ser mutation increased the tension threshold of the MSL8 channel while leaving conductance unchanged, and the Phe720Leu mutation severely disrupted the MSL8 channel. Both of these mutations impaired the ability of MSL8 to preserve pollen viability during hydration and to maintain the integrity of the pollen tube when expressed at endogenous levels. When overexpressed in an msl8-4 null background, MSL8I711S could partially rescue loss-of-function phenotypes, while MSL8F720L could not. When overexpressed in the wild-type Ler background, MSL8I711S suppressed pollen germination, similar to wild-type MSL8. In contrast, MSL8F720L failed to suppress pollen germination and increased pollen bursting, thereby phenocopying the msl8-4 mutant. Thus, an intact MSL8 channel is required for normal pollen function during hydration and germination. These data establish MSL8 as the first plant MS channel to fulfill previously established criteria for assignment as a mechanotransducer.

Is floral structure a reliable indicator of breeding system in the Brassicaceae?

This study investigated the usefulness of floral characters as a potential indicator of breeding system in the Brassicaceae. Initially, pod set, seed set and pollen tube growth experiments were carried out to confirm the breeding systems of 53 lines representing 25 different cultivated and weedy species from the Brassicaceae. The results of the pod set tests clearly differentiated between self-compatible and self-incompatible species. Floral characters were then evaluated on one or more lines of each of the 25 species. Fourteen floral characters were evaluated including, flower diameter, Cruden's outcrossing index, timing and direction of dehiscence and pollen-ovule ratio. Significant differences between species were evident in all of the floral characteristics evaluated. Flower diameter was generally larger in self-incompatible species than self-compatible species and pollen/ovule ratio was generally higher in self-incompatible species than self-compatible species. However, none of the floral characteristics was able to clearly differentiate the self-compatible and self-incompatible species and allow prediction of the breeding system with absolute confidence. The floral characteristic which was most effective at differentiating the two groups was anther direction at dehiscence.

Acetylglutamate kinase is required for both gametophyte function and embryo development in Arabidopsis thaliana.

The specific functions of the genes encoding arginine biosynthesis enzymes in plants are not well characterized. We report the isolation and characterization of Arabidopsis thaliana N-acetylglutamate kinase (NAGK), which catalyzes the second step of arginine biosynthesis. NAGK is a plastid-localized protein and is expressed during most developmental processes in Arabidopsis. Heterologous expression of the Arabidopsis NAGK gene in a NAGK-deficient Escherichia coli strain fully restores bacterial growth on arginine-deficient medium. nagk mutant pollen tubes grow more slowly than wild type pollen tubes and the phenotype is restored by either specifically through complementation by NAGK in pollen, or exogenous supplementation of arginine. nagk female gametophytes are defective in micropylar pollen tube guidance due to the fact that female gametophyte cell fate specification was specifically affected. Expression of NAGK in synergid cells rescues the defect of nagk female gametophytes. Loss-of-function of NAGK results in Arabidopsis embryos not developing beyond the four-celled embryo stage. The embryo-defective phenotype in nagk/NAGK plants cannot be rescued by watering nagk/NAGK plants with arginine or ornithine supplementation. In conclusion, our results reveal a novel role of NAGK and arginine in regulating gametophyte function and embryo development, and provide valuable insights into arginine transport during embryo development.