Poly(A) polymerase 1 contributes to competence acquisition of pollen tubes growing through the style in Arabidopsis thaliana.

Polyadenylation of mRNAs is critical for their export from the nucleus, stability, and efficient translation. The Arabidopsis thaliana genome encodes three isoforms of canonical nuclear poly(A) polymerase (PAPS) that redundantly polyadenylate the bulk of pre-mRNAs. However, previous studies have indicated that subsets of pre-mRNAs are preferentially polyadenylated by either PAPS1 or the other two isoforms. Such functional specialization raises the possibility of an additional level of gene-expression control in plants. Here we test this notion by studying the function of PAPS1 in pollen-tube growth and guidance. Pollen tubes growing through female tissue acquire the competence to find ovules efficiently and upregulate PAPS1 expression at the transcriptional, but not detectably at the protein level compared with in vitro grown pollen tubes. Using the temperature-sensitive paps1-1 allele we show that PAPS1 activity during pollen-tube growth is required for full acquisition of competence, resulting in inefficient fertilization by paps1-1 mutant pollen tubes. While these mutant pollen tubes grow almost at the wild-type rate, they are compromised in locating the micropyles of ovules. Previously identified competence-associated genes are less expressed in paps1-1 mutant than in wild-type pollen tubes. Estimating the poly(A) tail lengths of transcripts suggests that polyadenylation by PAPS1 is associated with reduced transcript abundance. Our results therefore suggest that PAPS1 plays a key role in the acquisition of competence and underline the importance of functional specialization between PAPS isoforms throughout different developmental stages.

Reproductive interference between alien species in Veronica.

Reproductive interference can lead to the exclusive distribution of species. Reports on reproductive interference between alien species are scarce, although alien species are becoming more abundant and evaluations of the interference between aliens and its influence on community structure are essential. We therefore investigated the presence of interference in two alien species of Veronica, V. cymbalaria and V. persica. Hand-pollination experiments revealed that heterospecific pollen adversely affected both species, but asymmetrically: in V. cymbalaria, it reduced the number of seeds both before and after conspecific pollination, but in V. persica it reduced the number of seeds only when it occurred before conspecific pollination. Field observation suggested that the number of seeds produced by V. cymbalaria was more influenced by the number of conspecific flowers than by the frequency of surrounding V. persica flowers. Pot experiments did not show a negative effect of heterospecific individuals on the reproductive success of focal species in either species. Observations of visiting pollinators revealed no pollinator preference between the plant species. Although we demonstrated asymmetrical reproductive interference between V. cymbalaria and V. persica, its effect in the field seemed limited. An essential factor mitigating the adverse effect of reproductive interference might be a shortage of pollinators that could facilitate interspecific pollination. We predict that further invasion of V. cymbalaria into areas where V. persica prevails is unlikely, although swift displacement of V. cymbalaria by V. persica in areas where V. cymbalaria is already established is also unlikely.

Comparisons among populations and individuals to evaluate pollen-pistil interaction as a mechanism of reproductive interference in Taraxacum.

Reproductive interference (RI), an interspecific mating interaction that reduces the fitness of at least one of the species involved, can lead to exclusive distributions in closely related species. A hypothesis previously proposed is that RI in plants may occur by ovule usurpation, in which pistils lack interspecific incompatibility and mistakenly accept heterospecific pollen, thereby losing an opportunity for conspecific pollen fertilization. However, few comparative studies have evaluated the consistency of the inferred mechanism within and among individuals and populations. We conducted hand-pollination experiments in six populations of three native Taraxacum species that suffered from different levels of RI from an alien congener, T. officinale, and compared pollen-pistil interactions among populations. We also investigated the interactions for eight individual T. japonicum plants whose response to heterospecific pollen deposition had been previously measured. Our results revealed that pollen tubes often penetrated native ovaries following heterospecific pollination in populations suffering from strong RI, whereas they seldom did in populations suffering from marginal RI. However, the relative frequency of the pollen tube penetration was not significantly related to the strength of alien RI. Not all pistils on an individual plant showed the same pollen receptivity following heterospecific pollination; rather, some accepted and some refused the pollen tubes. The relationship between pollen tube penetration following heterospecific pollination and the strength of the alien RI was also not significant among individuals. Our present results generally support the ovule usurpation hypothesis, but suggest that other factors, such as competition for pollinator services, variation in the effects of heterospecific pollen donors, and condition of the native inflorescences, might also affect the observed RI strength.

KOMPEITO, an Atypical Arabidopsis Rhomboid-Related Gene, Is Required for Callose Accumulation and Pollen Wall Development.

Fertilization is a key event for sexually reproducing plants. Pollen-stigma adhesion, which is the first step in male-female interaction during fertilization, requires proper pollen wall patterning. Callose, which is a ß-1.3-glucan, is an essential polysaccharide that is required for pollen development and pollen wall formation. Mutations in CALLOSE SYNTHASE 5 (CalS5) disrupt male meiotic callose accumulation; however, how CalS5 activity and callose synthesis are regulated is not fully understood. In this paper, we report the isolation of a kompeito-1 (kom-1) mutant defective in pollen wall patterning and pollen-stigma adhesion in Arabidopsis thaliana. Callose was not accumulated in kom-1 meiocytes or microspores, which was very similar to the cals5 mutant. The KOM gene encoded a member of a subclass of Rhomboid serine protease proteins that lacked active site residues. KOM was localized to the Golgi apparatus, and both KOM and CalS5 genes were highly expressed in meiocytes. A 220 kDa CalS5 protein was detected in wild-type (Col-0) floral buds but was dramatically reduced in kom-1. These results suggested that KOM was required for CalS5 protein accumulation, leading to the regulation of meiocyte-specific callose accumulation and pollen wall formation.

Fine-scale empirical data on niche divergence and homeolog expression patterns in an allopolyploid and its diploid progenitor species.

Polyploidization is pervasive in plants, but little is known about the niche divergence of wild allopolyploids (species that harbor polyploid genomes originating from different diploid species) relative to their diploid progenitor species and the gene expression patterns that may underlie such ecological divergence. We conducted a fine-scale empirical study on habitat and gene expression of an allopolyploid and its diploid progenitors. We quantified soil properties and light availability of habitats of an allotetraploid Cardamine flexuosa and its diploid progenitors Cardamine amara and Cardamine hirsuta in two seasons. We analyzed expression patterns of genes and homeologs (homeologous gene copies in allopolyploids) using RNA sequencing. We detected niche divergence between the allopolyploid and its diploid progenitors along water availability gradient at a fine scale: the diploids in opposite extremes and the allopolyploid in a broader range between diploids, with limited overlap with diploids at both ends. Most of the genes whose homeolog expression ratio changed among habitats in C. flexuosa varied spatially and temporally. These findings provide empirical evidence for niche divergence between an allopolyploid and its diploid progenitor species at a fine scale and suggest that divergent expression patterns of homeologs in an allopolyploid may underlie its persistence in diverse habitats.

Environmental sensing and morphological plasticity in plants.

All creatures on earth are affected by their surrounding environment. Animals can move and escape unfavorable environmental changes, whereas plants must respond to environmental stimuli. Plants adapt to changes with cellular-level responses to short-term environmental changes, but may adapt to changes in the environment by regulating their development and growth. In this review, we considered changes in atmospheric CO2 concentrations, dry/wet moisture conditions, flooding, and temperature as examples of environmental stimuli. We mainly focused on leaf morphology and stomatal density as examples of developmental and growth patterns of plants in response to the environment.

Direct interaction of ligand-receptor pairs specifying stomatal patterning.

Valves on the plant epidermis called stomata develop according to positional cues, which likely involve putative ligands (EPIDERMAL PATTERNING FACTORS [EPFs]) and putative receptors (ERECTA family receptor kinases and TOO MANY MOUTHS [TMM]) in Arabidopsis. Here we report the direct, robust, and saturable binding of bioactive EPF peptides to the ERECTA family. In contrast, TMM exhibits negligible binding to EPF1 but binding to EPF2. The ERECTA family forms receptor homomers in vivo. On the other hand, TMM associates with the ERECTA family but not with itself. While ERECTA family receptor kinases exhibit complex redundancy, blocking ERECTA and ERECTA-LIKE1 (ERL1) signaling confers specific insensitivity to EPF2 and EPF1, respectively. Our results place the ERECTA family as the primary receptors for EPFs with TMM as a signal modulator and establish EPF2-ERECTA and EPF1-ERL1 as ligand-receptor pairs specifying two steps of stomatal development: initiation and spacing divisions.

Comparison of mechanisms of reproductive interference in Taraxacum.

BACKGROUND AND AIMS: Reproductive interference may reduce fitness of either of the involved species, with potentially important ecological and evolutionary consequences. Except for the effect of shared pollinators on reproductive success, however, mechanisms underlying reproductive interference have been little studied, even though the severity of its impact may depend on the specific mechanism. The aim of this study was therefore to explore the mechanisms of reproductive interference between Taraxacum japonicum (native to Japan) and Taraxacum officinale (alien). METHODS: In a field survey, the association between alien species density and seed set in T. japonicum, and whether pollinator behaviour indicated a preference for the alien, were examined. Effects of heterospecific pollen deposition were measured in a series of hand pollination experiments, including mixed pollination experiments in which the order of application of conspecific and heterospecific pollen was varied. Finally, to investigate hybridization frequency, the parentage of seedlings produced following natural, mixed or heterospecific pollination was compared. KEY RESULTS: Alien species density did not negatively affect native seed set, nor did pollinators appear to have a preference for alien flowers. The hand pollination experiments showed that heterospecific pollen deposition adversely affected native seed set, especially when alien pollen was applied before conspecific pollen. No viable hybrids were found following natural pollination, which suggests that hybridization might be a rare event. CONCLUSION: Among the examined mechanisms, heterospecific pollen deposition might have the largest deleterious effect on the native species. This effect is frequency dependent; thus, a positive feedback loop may cause the effect on the population dynamics to increase over time, with the result that the alien might eventually displace the native in a population. Effects of the examined mechanisms on population dynamics should be investigated further to improve understanding of the impact of reproductive interference on the structure of plant communities.

Cell-cell communications and molecular mechanisms in plant sexual reproduction.

Sexual reproduction is achieved by precise interactions between male and female reproductive organs. In plant fertilization, sperm cells are carried to ovules by pollen tubes. Signals from the pistil are involved in elongation and control of the direction of the pollen tube. Genetic, reverse genetic, and cell biological analyses using model plants have identified various factors related to the regulation of pollen tube growth and guidance. In this review, I summarize the mechanisms and molecules controlling pollen tube growth to the ovule, micropylar guidance, reception of the guidance signal in the pollen tube, rupture of the pollen tube to release sperm cells, and cessation of the tube guidance signal. I also briefly introduce various techniques used to analyze pollen tube guidance in vitro.

Ca2+-activated reactive oxygen species production by Arabidopsis RbohH and RbohJ is essential for proper pollen tube tip growth.

In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca(2+) binding EF-hand motifs, possessed Ca(2+)-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca(2+)-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca(2+)-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca(2+). Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca(2+)-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.

Variation in the strength of reproductive interference from an alien congener to a native species in Taraxacum.

Reproductive interference (RI) may be a contributing factor to the displacement of native species by an alien congener, and RI strength has been shown theoretically to affect distributional relationships between species. Thus, variations in RI strength from alien to native species result in different consequences of invasions and efforts to conserve native species, but the variations have seldom been examined empirically. We therefore investigated RI strength variations from the alien species Taraxacum officinale and its hybrids to eight populations of native dandelions, four T. japonicum populations and two populations each of two subspecies of T. platycarpum. We examined the association between alien relative abundance and native seed set in field surveys, and we also performed hand-pollination experiments to investigate directly the sensitivity of native flowers to alien pollen. We found that the effect of alien relative abundance on native seed set of even the same native species could differ greatly in different regions, and that the sensitivity of native flowers to alien pollen was also dependent on region. Our results, together with those of previous studies, show that RI from the alien to the native species is strong in regions where the alien species outnumbers the native species and marginal where it does not; this result suggests that alien RI can critically affect distributional relationships between native and alien species. Our study highlights the importance of performing additional empirical investigations of RI strength variation and of giving due attention to alien RI in efforts to conserve regional native biodiversity.

Arabidopsis homeodomain-leucine zipper IV proteins promote stomatal development and ectopically induce stomata beyond the epidermis.

The shoot epidermis of land plants serves as a crucial interface between plants and the atmosphere: pavement cells protect plants from desiccation and other environmental stresses, while stomata facilitate gas exchange and transpiration. Advances have been made in our understanding of stomatal patterning and differentiation, and a set of 'master regulatory' transcription factors of stomatal development have been identified. However, they are limited to specifying stomatal differentiation within the epidermis. Here, we report the identification of an Arabidopsis homeodomain-leucine zipper IV (HD-ZIP IV) protein, HOMEODOMAIN GLABROUS2 (HDG2), as a key epidermal component promoting stomatal differentiation. HDG2 is highly enriched in meristemoids, which are transient-amplifying populations of stomatal-cell lineages. Ectopic expression of HDG2 confers differentiation of stomata in internal mesophyll tissues and occasional multiple epidermal layers. Conversely, a loss-of-function hdg2 mutation delays stomatal differentiation and, rarely but consistently, results in aberrant stomata. A closely related HD-ZIP IV gene, Arabidopsis thaliana MERISTEM LAYER1 (AtML1), shares overlapping function with HDG2: AtML1 overexpression also triggers ectopic stomatal differentiation in the mesophyll layer and atml1 mutation enhances the stomatal differentiation defects of hdg2. Consistently, HDG2 and AtML1 bind the same DNA elements, and activate transcription in yeast. Furthermore, HDG2 transactivates expression of genes that regulate stomatal development in planta. Our study highlights the similarities and uniqueness of these two HD-ZIP IV genes in the specification of protodermal identity and stomatal differentiation beyond predetermined tissue layers.

Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells.

For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules. However, there has been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Emerging data indicate that two synergid cells on the side of the egg cell emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen tube guidance. Here we report that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins are attractants derived from the synergid cells. We isolated synergid cells of Torenia fournieri, a unique plant with a protruding embryo sac, to identify transcripts encoding secreted proteins as candidate molecules for the chemoattractant(s). We found two CRPs, abundantly and predominantly expressed in the synergid cell, which are secreted to the surface of the egg apparatus. Moreover, they showed activity in vitro to attract competent pollen tubes of their own species and were named as LUREs. Injection of morpholino antisense oligomers against the LUREs impaired pollen tube attraction, supporting the finding that LUREs are the attractants derived from the synergid cells of T. fournieri.

Germline ß-1,3-glucan deposits are required for female gametogenesis in Arabidopsis thaliana.

Correct regulation of intercellular communication is a fundamental requirement for cell differentiation. In Arabidopsis thaliana, the female germline differentiates from a single somatic ovule cell that becomes encased in ß-1,3-glucan, a water insoluble polysaccharide implicated in limiting pathogen invasion, regulating intercellular trafficking in roots, and promoting pollen development. Whether ß-1,3-glucan facilitates germline isolation and development has remained contentious, since limited evidence is available to support a functional role. Here, transcriptional profiling of adjoining germline and somatic cells revealed differences in gene expression related to ß-1,3-glucan metabolism and signalling through intercellular channels (plasmodesmata). Dominant expression of a ß-1,3-glucanase in the female germline transiently perturbed ß-1,3-glucan deposits, allowed intercellular movement of tracer molecules, and led to changes in germline gene expression and histone marks, eventually leading to termination of germline development. Our findings indicate that germline ß-1,3-glucan fulfils a functional role in the ovule by insulating the primary germline cell, and thereby determines the success of downstream female gametogenesis.

Dysregulation of cell-to-cell connectivity and stomatal patterning by loss-of-function mutation in Arabidopsis chorus (glucan synthase-like 8).

Patterning of stomata, valves on the plant epidermis, requires the orchestrated actions of signaling components and cell-fate determinants. To understand the regulation of stomatal patterning, we performed a genetic screen using a background that partially lacks stomatal signaling receptors. Here, we report the isolation and characterization of chorus (chor), which confers excessive proliferation of stomatal-lineage cells mediated by SPEECHLESS (SPCH). chor breaks redundancy among three ERECTA family genes and strongly enhances stomatal patterning defects caused by loss-of-function in TOO MANY MOUTHS. chor seedlings also exhibit incomplete cytokinesis and growth defects, including disruptions in root tissue patterning and root hair cell morphogenesis. CHOR encodes a putative callose synthase, GLUCAN SYNTHASE-LIKE 8 (GSL8), that is required for callose deposition at the cell plate, cell wall and plasmodesmata. Consistently, symplastic macromolecular diffusion between epidermal cells is significantly increased in chor, and proteins that do not normally move cell-to-cell, including a fluorescent protein-tagged SPCH, diffuse to neighboring cells. Such a phenotype is not a general trait caused by cytokinesis defects. Our findings suggest that the restriction of symplastic movement might be an essential step for the proper segregation of cell-fate determinants during stomatal development.

Chemical synthesis of Torenia plant pollen tube attractant proteins by KAHA ligation.

The synthesis of secreted cysteine-rich proteins (CRPs) is a long-standing challenge due to protein aggregation and premature formation of inter- and intramolecular disulfide bonds. Chemical synthesis provides reduced CRPs with a higher purity, which is advantageous for folding and isolation. Herein, we report the chemical synthesis of pollen tube attractant CRPs Torenia fournieri LURE (TfLURE) and Torenia concolor LURE (TcLURE) and their chimeric analogues via α-ketoacid-hydroxylamine (KAHA) ligation. The bioactivity of chemically synthesized TfLURE protein was shown to be comparable to E. coli expressed recombinant protein through in vitro assay. The convergent protein synthesis approach is beneficial for preparing these small protein variants efficiently.

Identification and characterization of TcCRP1, a pollen tube attractant from Torenia concolor.

BACKGROUND AND AIMS: During sexual reproduction in higher angiosperms, the pollen tubes are directed to the ovules in the pistil to deliver sperm cells. This pollen tube attraction is highly species specific, and a group of small secreted proteins, TfCRPs, are necessary for this process in Torenia fournieri. METHODS: A candidate pollen tube attractant protein in Torenia concolor, a related species of T. fournieri, was isolated and the attractant abilities between them were compared. KEY RESULTS: TcCRP1, an orthologous gene of TfCRP1 from T. concolor, is expressed predominantly in the synergid cell. The gene product attracted pollen tubes in a concentration-dependent manner, but attracted fewer pollen tubes from the other species. CONCLUSIONS: The results indicated that this class of CRP proteins is a common pollen tube attractant in Torenia species. The sequence diversity of these proteins is important for species-specific pollen tube attraction.

Adaptive reduction of male gamete number in the selfing plant Arabidopsis thaliana.

The number of male gametes is critical for reproductive success and varies between and within species. The evolutionary reduction of the number of pollen grains encompassing the male gametes is widespread in selfing plants. Here, we employ genome-wide association study (GWAS) to identify underlying loci and to assess the molecular signatures of selection on pollen number-associated loci in the predominantly selfing plant Arabidopsis thaliana. Regions of strong association with pollen number are enriched for signatures of selection, indicating polygenic selection. We isolate the gene REDUCED POLLEN NUMBER1 (RDP1) at the locus with the strongest association. We validate its effect using a quantitative complementation test with CRISPR/Cas9-generated null mutants in nonstandard wild accessions. In contrast to pleiotropic null mutants, only pollen numbers are significantly affected by natural allelic variants. These data support theoretical predictions that reduced investment in male gametes is advantageous in predominantly selfing species.

Artificial selection reveals the role of transcriptional constraints in the maintenance of life history variation.

The trade-off between reproduction and self-maintenance is a cornerstone of life history theory, yet its proximate underpinnings are elusive. Here, we used an artificial selection approach to create replicated lines of Japanese quail (Coturnix japonica) that differ genetically in their reproductive investment. Whole transcriptome sequencing revealed that females from lines selected for high reproductive output show a consistent upregulation of genes associated with reproduction but a simultaneous downregulation of immune genes. Concordant phenotypic differences in immune function (i.e., specific antibody response against keyhole limpet hemocyanin) were observed between the selection lines, even in males who do not provide parental care. Our findings demonstrate the key role of obligate transcriptional constraints in the maintenance of life history variation. These constraints set fundamental limits to productivity and health in natural and domestic animal populations.