Synchronization of Arabidopsis flowering time and vegetative growth stage via FT overexpression can reveal inherent heterosis due to heterozygosity in intraspecific hybrids.

Intraspecific hybrids of Arabidopsis sometimes display heterosis. However, allelic variation of flowering repressor genes causes late flowering in F1, which might distort the potential heterosis effect due to prolonged vegetative growth. Here, overexpression of flowering gene FT synchronized flowering and eliminated growth differentials between parental and F1. These findings indicate the possibility of quantitatively demonstrating the inherent heterosis caused by heterozygosity.

Application of Green-enhanced Nano-lantern as a bioluminescent ratiometric indicator for measurement of Arabidopsis thaliana root apoplastic fluid pH.

Plant root absorbs water and nutrients from the soil, and the root apoplastic fluid (AF) is an important intermediate between cells and the surrounding environment. The acid growth theory suggests that an acidic AF is needed for cell wall expansion during root growth. However, technical limitations have precluded the quantification of root apoplastic fluid pH (AF-pH). Here, we used Green-enhanced Nano-lantern (GeNL), a chimeric protein of the luciferase NanoLuc (Nluc) and the green fluorescent protein mNeonGreen (mNG), as a ratiometric pH indicator based on the pH dependency of bioluminescence resonance energy transfer efficiency from Nluc to mNG. Luminescence spectrum of GeNL changed reciprocally from pH 4.5 to 7.5, with a pKa of 5.5. By fusing GeNL to a novel signal peptide from Arabidopsis thaliana Cellulase 1, we localised GeNL in A. thaliana AF. We visualised AF dynamics at subcellular resolution over 30 min and determined flow velocity in the maturation zone to be 0.97± 0.06 µm/s. We confirmed that the developing root AF is acidic in the pH range of 5.1-5.7, suggesting that the AF-pH is tightly regulated during root elongation. These results support the acid growth theory and provide evidence for AF-pH maintenance despite changes in ambient pH.

A novel petal up-regulated PhXTH7 promoter analysis in Petunia hybrida by using bioluminescence reporter gene.

Flower opening is an important phenomenon in plant that indicates the readiness of the flower for pollination leading to petal expansion and pigmentation. This phenomenon has great impact on crop yield, which makes researches of its mechanism attractive for both plant physiology study and agriculture. Gene promoters directing the expression in petal during the petal cell wall modification and expansion when flower opens could be a convenient tool to analyze or monitor gene expression targeting this event. However, there are no reports of isolated gene promoters that can direct gene expression in petal or petal limb during the rapid cell wall dynamics when the flower opens. Xyloglucan endotransglucosylase/hydrolase 7 (XTH7), a cell wall modifying enzyme, was reported having up-regulated gene expression in the petal of Arabidopsis thaliana and Petunia hybrida. In this study, we fused a 1,904 bp length P. hybrida XTH7 promoter with a gene encoding a bright bioluminescent protein (Green enhanced Nano-lantern) to report gene expression and observed petal up-regulated bioluminescence activity by means of a consumer-grade camera. More importantly, this novel promoter demonstrated up-regulated activity in the petal limb of P. hybrida matured flower during flower opening. P. hybrida XTH7 promoter would be a useful tool for flowering study, especially for petal expansion research during flower opening.

Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer.

Using the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.

Cullin1-P is an Essential Component of Non-Self Recognition System in Self-Incompatibility in Petunia.

Self-incompatibility (SI) in flowering plants is a genetic reproductive barrier to distinguish self- and non-self pollen to promote outbreeding. In Solanaceae, self-pollen is rejected by the ribonucleases expressed in the styles (S-RNases), via its cytotoxic function. On the other side, the male-determinant is the S-locus F-box proteins (SLFs) expressed in pollen. Multiple SLFs collaboratively detoxify non-self S-RNases, therefore, non-self recognition is the mode of self-/non-self discrimination in Solanaceae. It is considered that SLFs function as a substrate-recognition module of the Skp1-Cullin1-F-box (SCF) complex that inactivates non-self S-RNases via their polyubiquitination, which leads to degradation by 26S proteasome. In fact, PhSSK1 (Petunia hybrida SLF-interacting Skp1-like1) was identified as a specific component of SCFSLF and was shown to be essential for detoxification of S-RNase in Petunia However, different molecules are proposed as the candidate Cullin1, another component of SCFSLF, and there is as yet no definite conclusion. Here, we identified five Cullin1s from the expressed sequence tags (ESTs) derived from the male reproductive organ in Petunia Among them, only PhCUL1-P was co-immunoprecipitated with S7-SLF2. In vitro protein-binding assay suggested that PhSSK1 specifically forms a complex with PhCUL1-P in an SLF-dependent manner. Knockdown of PhCUL1-P suppressed fertility of transgenic pollen in cross-compatible pollination in the functional S-RNase-dependent manner. These results suggested that SCFSLF selectively uses PhCUL1-P. Phylogeny of Cullin1s indicates that CUL1-P is recruited into the SI machinery during the evolution of Solanaceae, suggesting that the SI components have evolved differently among species in Solanaceae and Rosaceae, despite both families sharing the S-RNase-based SI.

Gene duplication and genetic exchange drive the evolution of S-RNase-based self-incompatibility in Petunia.

Self-incompatibility (SI) systems in flowering plants distinguish self- and non-self pollen to prevent inbreeding. While other SI systems rely on the self-recognition between specific male- and female-determinants, the Solanaceae family has a non-self recognition system resulting in the detoxification of female-determinants of S-ribonucleases (S-RNases), expressed in pistils, by multiple male-determinants of S-locus F-box proteins (SLFs), expressed in pollen. It is not known how many SLF components of this non-self recognition system there are in Solanaceae species, or how they evolved. We identified 16-20 SLFs in each S-haplotype in SI Petunia, from a total of 168 SLF sequences using large-scale next-generation sequencing and genomic polymerase chain reaction (PCR) techniques. We predicted the target S-RNases of SLFs by assuming that a particular S-allele must not have a conserved SLF that recognizes its own S-RNase, and validated these predictions by transformation experiments. A simple mathematical model confirmed that 16-20 SLF sequences would be adequate to recognize the vast majority of target S-RNases. We found evidence of gene conversion events, which we suggest are essential to the constitution of a non-self recognition system and also contribute to self-compatible mutations.

Calcium signalling mediates self-incompatibility response in the Brassicaceae.

Self-incompatibility in the Brassicaceae is controlled by multiple haplotypes encoding the pollen ligand (S-locus protein 11, SP11, also known as S-locus cysteine-rich protein, SCR) and its stigmatic receptor (S-receptor kinase, SRK). A haplotype-specific interaction between SP11/SCR and SRK triggers the self-incompatibility response that leads to self-pollen rejection, but the signalling pathway remains largely unknown. Here we show that Ca(2+) influx into stigma papilla cells mediates self-incompatibility signalling. Using self-incompatible Arabidopsis thaliana expressing SP11/SCR and SRK, we found that self-pollination specifically induced an increase in cytoplasmic Ca(2+) ([Ca(2+)]cyt) in papilla cells. Direct application of SP11/SCR to the papilla cell protoplasts induced Ca(2+) increase, which was inhibited by D-(-)-2-amino-5-phosphonopentanoic acid (AP-5), a glutamate receptor channel blocker. An artificial increase in [Ca(2+)]cyt in papilla cells arrested wild-type (WT) pollen hydration. Treatment of papilla cells with AP-5 interfered with self-incompatibility, and Ca(2+) increase on the self-incompatibility response was reduced in the glutamate receptor-like channel (GLR) gene mutants. These results suggest that Ca(2+) influx mediated by GLR is the essential self-incompatibility response leading to self-pollen rejection.

Ubiquitin-proteasome-mediated degradation of S-RNase in a solanaceous cross-compatibility reaction.

Many plants have a self-incompatibility (SI) system in which the rejection of self-pollen is determined by multiple haplotypes at a single locus, termed S. In the Solanaceae, each haplotype encodes a single ribonuclease (S-RNase) and multiple S-locus F-box proteins (SLFs), which function as the pistil and pollen SI determinants, respectively. S-RNase is cytotoxic to self-pollen, whereas SLFs are thought to collaboratively recognize non-self S-RNases in cross-pollen and detoxify them via the ubiquitination pathway. However, the actual mechanism of detoxification remains unknown. Here we isolate the components of a SCF(SLF) (SCF = SKP1-CUL1-F-box-RBX1) from Petunia pollen. The SCF(SLF) polyubiquitinates a subset of non-self S-RNases in vitro. The polyubiquitinated S-RNases are degraded in the pollen extract, which is attenuated by a proteasome inhibitor. Our findings suggest that multiple SCF(SLF) complexes in cross-pollen polyubiquitinate non-self S-RNases, resulting in their degradation by the proteasome.

A pollen coat-inducible autoinhibited Ca2+-ATPase expressed in stigmatic papilla cells is required for compatible pollination in the Brassicaceae.

In the Brassicaceae, intraspecific non-self pollen (compatible pollen) can germinate and grow into stigmatic papilla cells, while self-pollen or interspecific pollen is rejected at this stage. However, the mechanisms underlying this selective acceptance of compatible pollen remain unclear. Here, using a cell-impermeant calcium indicator, we showed that the compatible pollen coat contains signaling molecules that stimulate Ca(2+) export from the papilla cells. Transcriptome analyses of stigmas suggested that autoinhibited Ca(2+)-ATPase13 (ACA13) was induced after both compatible pollination and compatible pollen coat treatment. A complementation test using a yeast Saccharomyces cerevisiae strain lacking major Ca(2+) transport systems suggested that ACA13 indeed functions as an autoinhibited Ca(2+) transporter. ACA13 transcription increased in papilla cells and in transmitting tracts after pollination. ACA13 protein localized to the plasma membrane and to vesicles near the Golgi body and accumulated at the pollen tube penetration site after pollination. The stigma of a T-DNA insertion line of ACA13 exhibited reduced Ca(2+) export, as well as defects in compatible pollen germination and seed production. These findings suggest that stigmatic ACA13 functions in the export of Ca(2+) to the compatible pollen tube, which promotes successful fertilization.

Cytoplasmic Ca2+ changes dynamically during the interaction of the pollen tube with synergid cells.

The directional growth of the pollen tube from the stigma to the embryo sac in the ovules is regulated by pollen-pistil interactions based on intercellular communication. Although pollen tube growth is regulated by the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)), it is not known whether [Ca(2+)](cyt) is involved in pollen tube guidance and reception. Using Arabidopsis expressing the GFP-based Ca(2+)-sensor yellow cameleon 3.60 (YC3.60) in pollen tubes and synergid cells, we monitored Ca(2+) dynamics in these cells during pollen tube guidance and reception under semi-in vivo fertilization conditions. In the pollen tube growing towards the micropyle, pollen tubes initiated turning within 150 µm of the micropylar opening; the [Ca(2+)](cyt) in these pollen tube tips was higher than in those not growing towards an ovule in assays with myb98 mutant ovules, in which pollen tube guidance is disrupted. These results suggest that attractants secreted from the ovules affect Ca(2+) dynamics in the pollen tube. [Ca(2+)](cyt) in synergid cells did not change when the pollen tube grew towards the micropyle or entered the ovule. Upon pollen tube arrival at the synergid cell, however, [Ca(2+)](cyt) oscillation began at the micropylar pole of the synergid, spreading towards the chalazal pole. Finally, [Ca(2+)](cyt) in the synergid cell reached a maximum at pollen tube rupture. These results suggest that signals from the pollen tube induce Ca(2+) oscillations in synergid cells, and that this Ca(2+) oscillation is involved in the interaction between the pollen tube and synergid cell.

Collaborative non-self recognition system in S-RNase-based self-incompatibility.

Self-incompatibility in flowering plants prevents inbreeding and promotes outcrossing to generate genetic diversity. In Solanaceae, a multiallelic gene, S-locus F-box (SLF), was previously shown to encode the pollen determinant in self-incompatibility. It was postulated that an SLF allelic product specifically detoxifies its non-self S-ribonucleases (S-RNases), allelic products of the pistil determinant, inside pollen tubes via the ubiquitin-26S-proteasome system, thereby allowing compatible pollinations. However, it remained puzzling how SLF, with much lower allelic sequence diversity than S-RNase, might have the capacity to recognize a large repertoire of non-self S-RNases. We used in vivo functional assays and protein interaction assays to show that in Petunia, at least three types of divergent SLF proteins function as the pollen determinant, each recognizing a subset of non-self S-RNases. Our findings reveal a collaborative non-self recognition system in plants.

Fine-tuning of the cytoplasmic Ca2+ concentration is essential for pollen tube growth.

Pollen tube growth is crucial for the delivery of sperm cells to the ovule during flowering plant reproduction. Previous in vitro imaging of Lilium longiflorum and Nicotiana tabacum has shown that growing pollen tubes exhibit a tip-focused Ca(2+) concentration ([Ca(2+)]) gradient and regular oscillations of the cytosolic [Ca(2+)] ([Ca(2+)](cyt)) in the tip region. Whether this [Ca(2+)] gradient and/or [Ca(2+)](cyt) oscillations are present as the tube grows through the stigma (in vivo condition), however, is still not clear. We monitored [Ca(2+)](cyt) dynamics in pollen tubes under various conditions using Arabidopsis (Arabidopsis thaliana) and N. tabacum expressing yellow cameleon 3.60, a fluorescent calcium indicator with a large dynamic range. The tip-focused [Ca(2+)](cyt) gradient was always observed in growing pollen tubes. Regular oscillations of the [Ca(2+)](cyt), however, were rarely identified in Arabidopsis or N. tabacum pollen tubes grown under the in vivo condition or in those placed in germination medium just after they had grown through a style (semi-in vivo condition). On the other hand, regular oscillations were observed in vitro in both growing and nongrowing pollen tubes, although the oscillation amplitude was 5-fold greater in the nongrowing pollen tubes compared with growing pollen tubes. These results suggested that a submicromolar [Ca(2+)](cyt) in the tip region is essential for pollen tube growth, whereas a regular [Ca(2+)] oscillation is not. Next, we monitored [Ca(2+)] dynamics in the endoplasmic reticulum ([Ca(2+)](ER)) in relation to Arabidopsis pollen tube growth using yellow cameleon 4.60, which has a lower affinity for Ca(2+) compared with yellow cameleon 3.60. The [Ca(2+)](ER) in pollen tubes grown under the semi-in vivo condition was between 100 and 500 microm. In addition, cyclopiazonic acid, an inhibitor of ER-type Ca(2+)-ATPases, inhibited growth and decreased the [Ca(2+)](ER). Our observations suggest that the ER serves as one of the Ca(2+) stores in the pollen tube and cyclopiazonic acid-sensitive Ca(2+)-ATPases in the ER are required for pollen tube growth.

Design of peptide inhibitors for the importin alpha/beta nuclear import pathway by activity-based profiling.

Despite the current availability of selective inhibitors for the classical nuclear export pathway, no inhibitor for the classical nuclear import pathway has been developed. Here we describe the development of specific inhibitors for the importin alpha/beta pathway using a novel method of peptide inhibitor design. An activity-based profile was created via systematic mutational analysis of a peptide template of a nuclear localization signal. An additivity-based design using the activity-based profile generated two peptides with affinities for importin alpha that were approximately 5 million times higher than that of the starting template sequence. The high affinity of these peptides resulted in specific inhibition of the importin alpha/beta pathway. These peptide inhibitors provide a useful tool for studying nuclear import events. Moreover, our inhibitor design method should enable the development of potent inhibitors from a peptide seed.

Actin dynamics in papilla cells of Brassica rapa during self- and cross-pollination.

The self-incompatibility system of the plant species Brassica is controlled by the S-locus, which contains S-RECEPTOR KINASE (SRK) and S-LOCUS PROTEIN11 (SP11). SP11 binding to SRK induces SRK autophosphorylation and initiates a signaling cascade leading to the rejection of self pollen. However, the mechanism controlling hydration and germination arrest during self-pollination is unclear. In this study, we examined the role of actin, a key cytoskeletal component regulating the transport system for hydration and germination in the papilla cell during pollination. Using rhodamine-phalloidin staining, we showed that cross-pollination induced actin polymerization, whereas self-pollination induced actin reorganization and likely depolymerization. By monitoring transiently expressed green fluorescent protein fused to the actin-binding domain of mouse talin, we observed the concentration of actin bundles at the cross-pollen attachment site and actin reorganization and likely depolymerization at the self-pollen attachment site; the results correspond to those obtained by rhodamine-phalloidin staining. We further showed that the coat of self pollen is sufficient to mediate this response. The actin-depolymerizing drug cytochalasin D significantly inhibited pollen hydration and germination during cross-pollination, further emphasizing a role for actin in these processes. Additionally, three-dimensional electron microscopic tomography revealed the close association of the actin cytoskeleton with an apical vacuole network. Self-pollination disrupted the vacuole network, whereas cross-pollination led to vacuolar rearrangements toward the site of pollen attachment. Taken together, our data suggest that self- and cross-pollination differentially affect the dynamics of the actin cytoskeleton, leading to changes in vacuolar structure associated with hydration and germination.

Characterization of the SP11/SCR high-affinity binding site involved in self/nonself recognition in brassica self-incompatibility.

In Brassica self-incompatibility, the recognition of self/nonself pollen grains, is controlled by the S-locus, which encodes three highly polymorphic proteins: S-locus receptor kinase (SRK), S-locus protein 11 (SP11; also designated S-locus Cys-rich protein), and S-locus glycoprotein (SLG). SP11, located in the pollen coat, determines pollen S-haplotype specificity, whereas SRK, located on the plasma membrane of stigmatic papilla cells, determines stigmatic S-haplotype specificity. SLG shares significant sequence similarity with the extracellular domain of SRK and is abundant in the stigmatic cell wall, but its function is controversial. We previously showed that SP11 binds directly to its cognate SRK with high affinity (K(d) = 0.7 nM) and induces its autophosphorylation. We also found that an SLG-like, 60-kD protein on the stigmatic membrane forms a high-affinity binding site for SP11. Here, we show that the 60-kD stigmatic membrane protein is a truncated form of SRK containing the extracellular domain, transmembrane domain, and part of the juxtamembrane domain. A transiently expressed, membrane-anchored form of SRK exhibits high-affinity binding to SP11, whereas the soluble SRK (eSRK) lacking the transmembrane domain exhibits no high-affinity binding, as is the case with SLG. The different binding affinities of the membrane-anchored SRK and soluble eSRK or SLG will be significant for the specific perception of SP11 by SRK.

Calcium crystals in the anther of Petunia: the existence and biological significance in the pollination process.

Using an X-ray microanalysis system fitted with variable-pressure scanning electron microscopy, we noted that many calcium crystals accumulated under the stomium in the anther of Petunia. When the anther was dehisced and pollen grains were released from the stomata, the calcium crystals adhered to pollen grains and moved to the stigma together with pollen grains. In contrast, an X-ray microanalysis of the stigma surface before pollination detected no calcium emission on the stigma surface. Furthermore, pollen germination and pollen tube growth in medium without Ca occurred as in complete medium. However, after the pollen grains had been washed with abundant germination medium without calcium, pollen germination in the medium without Ca was inhibited. These results show that the calcium crystals dissolved in the aqueous drop under the exudate on the stigma and supplied calcium ions for pollen germination. In addition, calcium crystals were produced not only in the anther of Petunia but also in Nicotiana, suggesting that calcium crystals supply pollen grains with the calcium ions required for pollen germination and serve to improve reproduction efficiency in Solanaceae.

Comparative analysis of the self-incompatibility (S-) locus region of Prunus mume: identification of a pollen-expressed F-box gene with allelic diversity.

BACKGROUND: Self-incompatibility (SI) in the Solanaceae, Rosaceae and Scrophulariaceae is gametophytically controlled by a single polymorphic locus, termed the S-locus. To date, the only known S-locus product is a polymorphic ribonuclease, termed S-RNase, which is secreted by stylar tissue and thought to act as a cytotoxin that degrades the RNA of incompatible pollen tubes. However, understanding how S-RNase causes S-haplotype specific inhibition of pollen tubes has been hampered by the lack of a cloned pollen S-determinant gene. RESULTS: To identify the pollen S-determinant gene, we investigated the genomic structure of the S-locus region of the S1- and S7-haplotypes of Prunus mume (Japanese apricot), and identified 13 genes around the S-RNase gene. Among them, only one F-box gene, termed SLF (S-locus F-box), fulfilled the conditions for a pollen S-determinant gene: (i) together with the S-RNase gene, it is located within the highly divergent genomic region of the S-locus, (ii) it exhibits S-haplotype specific diversity among three analysed S-haplotypes, and (iii) it is specifically expressed in pollen, but not in the styles or leaves. CONCLUSION: The results indicate that SLF is a prime candidate for the pollen S-determinant gene of SI.

The dominance of alleles controlling self-incompatibility in Brassica pollen is regulated at the RNA level.

Self-incompatibility (SI) in Brassica is controlled sporophytically by the multiallelic S-locus. The SI phenotype of pollen in an S-heterozygote is determined by the relationship between the two S-haplotypes it carries, and dominant/recessive relationships often are observed between the two S-haplotypes. The S-locus protein 11 (SP11, also known as the S-locus cysteine-rich protein) gene has been cloned from many pollen-dominant S-haplotypes (class I) and shown to encode the pollen S-determinant. However, SP11 from pollen-recessive S-haplotypes (class II) has never been identified by homology-based cloning strategies, and how the dominant/recessive interactions between the two classes occur was not known. We report here the identification and molecular characterization of SP11s from six class II S-haplotypes of B. rapa and B. oleracea. Phylogenetic analysis revealed that the class II SP11s form a distinct group separated from class I SP11s. The promoter sequences and expression patterns of SP11s also were different between the two classes. The mRNA of class II SP11, which was detected predominantly in the anther tapetum in homozygotes, was not detected in the heterozygotes of class I and class II S-haplotypes, suggesting that the dominant/recessive relationships of pollen are regulated at the mRNA level of SP11s.