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.     

Electrostatic potentials of the S‐locus F‐box proteins contribute to the pollen S specificity in self‐incompatibility in Petunia hybrida

Self‐incompatibility (SI) is a self/non‐self discrimination system found widely in angiosperms and, in many species, is controlled by a single polymorphic S‐locus. In the Solanaceae, Rosaceae and Plantaginaceae, the S‐locus encodes a single S‐RNase and a cluster of S‐locus F‐box (SLF) proteins to control the pistil and pollen expression of SI, respectively. Previous studies have shown that their cytosolic interactions determine their recognition specificity, but the physical force between their interactions remains unclear. In this study, we show that the electrostatic potentials of SLF contribute to the pollen S specificity through a physical mechanism of ‘like charges repel and unlike charges attract’ between SLFs and S‐RNases in Petunia hybrida. Strikingly, the alteration of a single C‐terminal amino acid of SLF reversed its surface electrostatic potentials and subsequently the pollen S specificity. Collectively, our results reveal that the electrostatic potentials act as a major physical force between cytosolic SLFs and S‐RNases, providing a mechanistic insight into the self/non‐self discrimination between cytosolic proteins in angiosperms.     

The Skp1‐like protein SSK1 is required for cross‐pollen compatibility in S‐RNase‐based self‐incompatibility

The self‐incompatibility (SI) response occurs widely in flowering plants as a means of preventing self‐fertilization. In these self/non‐self discrimination systems, plant pistils reject self or genetically related pollen. In the Solanaceae, Plantaginaceae and Rosaceae, pistil‐secreted S‐RNases enter the pollen tube and function as cytotoxins to specifically arrest self‐pollen tube growth. Recent studies have revealed that the S‐locus F‐box (SLF) protein controls the pollen expression of SI in these families. However, the precise role of SLF remains largely unknown. Here we report that PhSSK1 (Petunia hybrida SLF‐interacting Skp1‐like1), an equivalent of AhSSK1 of Antirrhinum hispanicum, is expressed specifically in pollen and acts as an adaptor in an SCF(Skp1‐Cullin1‐F‐box)^SLF complex, indicating that this pollen‐specific SSK1‐SLF interaction occurs in both Petunia and Antirrhinum, two species from the Solanaceae and Plantaginaceae, respectively. Substantial reduction of PhSSK1 in pollen reduced cross‐pollen compatibility (CPC) in the S‐RNase‐based SI response, suggesting that the pollen S determinant contributes to inhibiting rather than protecting the S‐RNase activity, at least in solanaceous plants. Furthermore, our results provide an example that a specific Skp1‐like protein other than the known conserved ones can be recruited into a canonical SCF complex as an adaptor.     

Apple MdABCF assists in the transportation of S‐RNase into pollen tubes

Self‐incompatibility (SI) is a reproductive isolation mechanism in flowering plants. Plants in the Solanaceae, Rosaceae and Plantaginaceae belong to the gametophytic self‐incompatibility type. S‐RNase, which is encoded by a female‐specific gene located at the S locus, degrades RNA in the pollen tube and causes SI. Recent studies have provided evidence that S‐RNase is transported non‐selectively into the pollen tube, but have not specified how this transportation is accomplished. We show here that the apple (Malus domestica) MdABCF protein, which belongs to group F of the ABC transporter family, assists in transportation of S‐RNase into the pollen tube. MdABCF is located in the pollen tube membrane and interacts with S‐RNase. S‐RNase was unable to enter the pollen tube when MdABCF was silenced by antisense oligonucleotide transfection. Our results show that MdABCF assists in transportation of either self or non‐self S‐RNase into the pollen tube. Moreover, MdABCF coordinates with the cytoskeleton to transport S‐RNase. Blockage of S‐RNase transport disrupts self‐incompatibility in this system.     

A farnesyl pyrophosphate synthase gene expressed in pollen functions in S‐RNase‐independent unilateral incompatibility

Multiple independent and overlapping pollen rejection pathways contribute to unilateral interspecific incompatibility (UI). In crosses between tomato species, pollen rejection usually occurs when the female parent is self‐incompatible (SI) and the male parent self‐compatible (SC) (the ‘SI × SC rule’). Additional, as yet unknown, UI mechanisms are independent of self‐incompatibility and contribute to UI between SC species or populations. We identified a major quantitative trait locus on chromosome 10 (ui10.1) which affects pollen‐side UI responses in crosses between cultivated tomato, Solanum lycopersicum, and Solanum pennelliiLA0716, both of which are SC and lack S‐RNase, the pistil determinant of S‐specificity in Solanaceae. Here we show that ui10.1 is a farnesyl pyrophosphate synthase gene (FPS2) expressed in pollen. Expression is about 18‐fold higher in pollen of S. pennellii than in S. lycopersicum. Pollen with the hypomorphic S. lycopersicum allele is selectively eliminated on pistils of the F₁ hybrid, leading to transmission ratio distortion in the F₂ progeny. CRISPR/Cas9‐generated knockout mutants (fps2) in S. pennelliiLA0716 are self‐sterile due to pollen rejection, but mutant pollen is fully functional on pistils of S. lycopersicum. F₂ progeny of S. lycopersicum × S. pennellii (fps2) show reversed transmission ratio distortion due to selective elimination of pollen bearing the knockout allele. Overexpression of FPS2 in S. lycopersicum pollen rescues the pollen elimination phenotype. FPS2‐based pollen selectivity does not involve S‐RNase and has not been previously linked to UI. Our results point to an entirely new mechanism of interspecific pollen rejection in plants.     

HT proteins contribute to S‐RNase‐independent pollen rejection in Solanum

Plants have mechanisms to recognize and reject pollen from other species. Although widespread, these mechanisms are less well understood than the self‐incompatibility (SI) mechanisms plants use to reject pollen from close relatives. Previous studies have shown that some interspecific reproductive barriers (IRBs) are related to SI in the Solanaceae. For example, the pistil SI proteins S‐RNase and HT protein function in a pistil‐side IRB that causes rejection of pollen from self‐compatible (SC) red/orange‐fruited species in the tomato clade. However, S‐RNase‐independent IRBs also clearly contribute to rejecting pollen from these species. We investigated S‐RNase‐independent rejection of Solanum lycopersicum pollen by SC Solanum pennellii LA0716, SC. Solanum habrochaites LA0407, and SC Solanum arcanum LA2157, which lack functional S‐RNase expression. We found that all three accessions express HT proteins, which previously had been known to function only in conjunction with S‐RNase, and then used RNAi to test whether they also function in S‐RNase‐independent pollen rejection. Suppressing HT expression in SC S. pennellii LA0716 allows S. lycopersicum pollen tubes to penetrate farther into the pistil in HT suppressed plants, but not to reach the ovary. In contrast, suppressing HT expression in SC. Solanum habrochaites LA0407 and in SC S. arcanum LA2157 allows S. lycopersicum pollen tubes to penetrate to the ovary and produce hybrids that, otherwise, would be difficult to obtain. Thus, HT proteins are implicated in both S‐RNase‐dependent and S‐RNase‐independent pollen rejection. The results support the view that overall compatibility results from multiple pollen–pistil interactions with additive effects.     

Pollen tube growth in the self‐compatible sweet cherry genotype, ‘Cristobalina’, is slowed down after self‐pollination

Sweet cherry is a self‐incompatible fruit tree species in the Rosaceae. As other species in the family, sweet cherry exhibits S‐RNase‐based gametophytic self‐incompatibility. This mechanism is genetically determined by the S‐locus that encodes the pollen and pistil determinants, SFB and S‐RNase, respectively. Several self‐compatible sweet cherry genotypes have been described and most of them have mutations at the S‐locus leading to self‐compatibility. However, ‘Cristobalina’ sweet cherry is self‐compatible due to a mutation in a pollen function modifier that is not linked to the S‐locus. To investigate the physiology of self‐compatibility in this cultivar, S‐locus segregation in crosses involving ‘Cristobalina’ pollen, and pollen tube growth in self‐ and cross‐pollinations, were studied. In the crosses with genotypes sharing only one S‐haplotype, the non‐self S‐haplotype was inherited more frequently than the self S‐haplotype. Pollen tube growth studies revealed that the time to travel the whole length of the style was longer for self‐pollen tubes than for cross‐pollen tubes. Together, these results suggest that ‘Cristobalina’ pollen tube growth is slower after self‐pollination than after cross‐pollination. This reproductive strategy would allow self‐fertilisation in the absence of compatible pollen but would promote cross‐fertilisation if cross‐compatible pollen is available, a possible case of cryptic self‐incompatibility. This bet‐hedging strategy might be advantageous for an ecotype that is native to the mountains of the Spanish Mediterranean coast, in the geographical limits of the distribution of this species. ‘Cristobalina’ blooming takes place very early in the season when mating possibilities are scarce and, consequently, self‐compatibility may be the only possibility for this genotype to produce offspring.     

Apple S‐RNase interacts with an actin‐binding protein,MdMVG, to reduce pollen tube growth by inhibiting its actin‐severing activity at the early stage of self‐pollination induction

In S‐RNase‐mediated self‐incompatibility, S‐RNase secreted from the style destroys the actin cytoskeleton of the self‐pollen tubes, eventually halting their growth, but the mechanism of this process remains unclear. In vitro biochemical assays revealed that S‐RNase does not bind or sever filamentous actin (F‐actin). In apple (Malus domestica), we identified an actin‐binding protein containing myosin, villin and GRAM (MdMVG), that physically interacts with S‐RNase and directly binds and severs F‐actin. Immunofluorescence assays and total internal reflection fluorescence microscopy indicated that S‐RNase inhibits the F‐actin‐severing activity of MdMVG in vitro. In vivo, the addition of S‐RNase to self‐pollen tubes increased the fluorescence intensity of actin microfilaments and reduced the severing frequency of microfilaments and the rate of pollen tube growth in self‐pollination induction in the presence of MdMVG overexpression. By generating 25 single‐, double‐ and triple‐point mutations in the amino acid motif E‐E‐K‐E‐K of MdMVG via mutagenesis and testing the resulting mutants with immunofluorescence, we identified a triple‐point mutant, MdMVG^{(E167A/E171A/K185A)}, that no longer has F‐actin‐severing activity or interacts with any of the four S‐haplotype S‐RNases, indicating that all three amino acids (E167, E171 and K185) are essential for the severing activity of MdMVG and its interaction with S‐RNases. We conclude that apple S‐RNase interacts with MdMVG to reduce self‐pollen tube growth by inhibiting its F‐actin‐severing activity.     

NaTrxh is an essential protein for pollen rejection in Nicotiana by increasing S‐RNase activity

In self‐incompatible Solanaceae, the pistil protein S‐RNase contributes to S‐specific pollen rejection in conspecific crosses, as well as to rejecting pollen from foreign species or whole clades. However, S‐RNase alone is not sufficient for either type of pollen rejection. We describe a thioredoxin (Trx) type h from Nicotiana alata, NaTrxh, which interacts with and reduces S‐RNase in vitro. Here, we show that expressing a redox‐inactive mutant, NaTrxh_SS, suppresses both S‐specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia. Biochemical experiments provide evidence that NaTrxh specifically reduces the Cys₁₅₅‐Cys₁₈₅ disulphide bond of S_C10‐Rnase, resulting in a significant increase of its ribonuclease activity. This reduction and increase in S‐RNase activity by NaTrxh helps to explain why S‐RNase alone could be insufficient for pollen rejection.     

Paternal‐specific S‐allele transmission in sweet cherry (Prunus avium L.): the potential for sexual selection

Homomorphic self‐incompatibility is a well‐studied example of a physiological process that is thought to increase population diversity and reduce the expression of inbreeding depression. Whereas theoretical models predict the presence of a large number of S‐haplotypes with equal frequencies at equilibrium, unequal allele frequencies have been repeatedly reported and attributed to sampling effects, population structure, demographic perturbation, sheltered deleterious mutations or selection pressure on linked genes. However, it is unclear to what extent unequal segregations are the results of gametophytic or sexual selection. Although these two forces are difficult to disentangle, testing S‐alleles in the offspring of controlled crosses provides an opportunity to separate these two phenomena. In this work, segregation and transmission of S‐alleles have been characterized in progenies of mixed donors and fully compatible pollinations under field conditions in Prunus avium. Seed set patterns and pollen performance have also been characterized. The results reveal paternal‐specific distorted transmission of S‐alleles in most of the crosses. Interestingly, S‐allele segregation within any given paternal or maternal S‐locus was random. Observations on pollen germination, pollen tube growth rate, pollen tube cohort size, seed set dynamics and transmission patterns strongly suggest post‐pollination, prezygotic sexual selection, with male–male competition as the most likely mechanism. According to these results, post‐pollination sexual selection takes precedence over frequency‐dependent selection in explaining unequal S‐haplotype frequencies.     

Identification of differentially accumulating pistil proteins associated with self‐incompatibility of non‐heading Chinese cabbage

Non‐heading Chinese cabbage (Brassica campestris L. ssp. chinensis Makino), an important vegetable crop in China, exhibits a typical sporophytic self‐incompatibility (SI) system. To better understand the mechanism of SI response and identify potential candidate proteins involved in the SI system of this vegetable crop, the proteomic approach was taken to identify differential accumulating pistil proteins. Pistils were collected at 0 h and 2 h after self‐pollination at anthesis in self‐incompatible and compatible lines of non‐heading Chinese cabbage, and total proteins were extracted and separated by two‐dimensional gel electrophoresis (2‐DE). A total of 25 protein spots that displayed differential abundance were identified by matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometry (MALDI–TOF/TOF MS) and peptide mass fingerprinting (PMF). Among them, 22 protein spots were confidently established. The mRNA levels of the corresponding genes were detected by quantitative RT‐PCR. The 22 identified protein spots are involved in energy metabolism (four), protein biosynthesis (three), photosynthesis (six), stress response and defence (five), and protein degradation (four). Among these potential candidate proteins, UDP‐sugar pyrophosphorylase could be involved in sucrose degradation to influence pollen germination and growth. Glutathione S–transferases could be involved in pollen maturation, and affect pollen fertility. Senescence‐associated cysteine protease, which is related to programmed cell death, could be mainly related to self pollen recognition of non‐heading Chinese cabbage. The study will contribute to further investigations of molecular mechanism of sporophytic SI in Brassicaceae.     

Mitochondrial–nuclear co‐evolution leads to hybrid incompatibility through pentatricopeptide repeat proteins

Mitochondrial–nuclear incompatibility has a major role in reproductive isolation between species. However, the underlying mechanism and driving force of mitochondrial–nuclear incompatibility remain elusive. Here, we report a pentatricopeptide repeat‐containing (PPR) protein, Ccm1, and its interacting partner, 15S rRNA, to be involved in hybrid incompatibility between two yeast species, Saccharomyces cerevisiae and Saccharomyces bayanus. S. bayanus‐Ccm1 has reduced binding affinity for S. cerevisiae‐15S rRNA, leading to respiratory defects in hybrid cells. This incompatibility can be rescued by single mutations on several individual PPR motifs, demonstrating the highly evolvable nature of PPR proteins. When we examined other PPR proteins in the closely related Saccharomyces sensu stricto yeasts, about two‐thirds of them showed detectable incompatibility. Our results suggest that fast co‐evolution between flexible PPR proteins and their mitochondrial RNA substrates may be a common driving force in the development of mitochondrial–nuclear hybrid incompatibility.     

Two neutral variants segregating at the gametophytic self‐incompatibility locus of European pear (Pyrus communis L.) (Rosaceae,Pyrinae)

Extensive survey of the S‐locus diversity of plant species with RNase‐based gametophytic self‐incompatibility has failed to identify neutral variation segregating within S‐allele specificities. Although this is the expected result according to population genetics theory, it conflicts with recent models of S‐allele evolution, which suggest that new specificities might arise by a continuous process of subtle changes that individually do not alter the specificity of the S‐genes, but whose cumulative effects result in new S‐allele functions. Genomic analysis of S‐RNase sequences associated with the S₁₀₄ (=S₄, =S_b) allele of European pear (Pyrus communis L.) cultivars yielded two distinct variants (named herein S_104‐1 and S_104‐2) that differed at five nucleotide positions within the open reading frame, two of which resulted in changes in the predicted protein sequence. Test‐cross experiments indicated that the S‐alleles associated with the S_104‐1 and S_104‐2RNases exhibit the same pollen and pistil functions, suggesting that they are two neutral variants segregating within the S₁₀₄ haplotype of European pear. These allelic forms might represent transitional states in the process of generating new specificities in the species, in accordance with models that predict S‐function transition through neutral intermediates. This possibility was further evaluated through the pattern of molecular evolution of functionally distinct European pear S‐RNases, which indicated that most recent S‐allele diversification in this species proceeded in the absence of adaptive selective pressure.     

A COP1‐PIF‐HEC regulatory module fine‐tunes photomorphogenesis in Arabidopsis

Light responses mediated by the photoreceptors play crucial roles in regulating different aspects of plant growth and development. An E3 ubiquitin ligase complex CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)1/SUPPRESSOR OF PHYA (SPA), one of the central repressors of photomorphogenesis, is critical for maintaining skotomorphogenesis. It targets several positive regulators of photomorphogenesis for degradation in darkness. Recently, we revealed that basic helix‐loop‐helix factors, HECATEs (HECs), function as positive regulators of photomorphogenesis by directly interacting and antagonizing the activity of another group of repressors called PHYTOCHROME‐INTERACTING FACTORs (PIFs). It was also shown that HECs are partially degraded in the dark through the ubiquitin/26S proteasome pathway. However, the underlying mechanism of HEC degradation in the dark is still unclear. Here, we show that HECs also interact with both COP1 and SPA proteins in darkness, and that HEC2 is directly targeted by COP1 for degradation via the ubiquitin/26S proteasome pathway. Moreover, COP1‐mediated polyubiquitylation and degradation of HEC2 are enhanced by PIF1. Therefore, the ubiquitylation and subsequent degradation of HECs are significantly reduced in both cop1 and pif mutants. Consistent with this, the hec mutants partially suppress photomorphogenic phenotypes of both cop1 and pifQ mutants. Collectively, our work reveals that the COP1/SPA‐mediated ubiquitylation and degradation of HECs contributes to the coordination of skoto/photomorphogenic development in plants.     

Identification of the self-incompatibility locus F-box protein-containing complex in Petunia inflata

The polymorphic S-locus regulating self-incompatibility (SI) in Petunia contains the S-RNase gene and a number of S-locus F-box (SLF) genes. While penetrating the style through the stigma, a pollen tube takes up all S-RNases, but only self S-RNase inhibits pollen tube growth. Recent evidence suggests that SLFs produced by pollen collectively interact with and detoxify non-self S-RNases, but none can interact with self S-RNase. An SLF may be the F-box protein component of an SCF complex (containing Cullin1, Skp1 and Rbx1), which mediates ubiquitination of protein substrates for degradation by the 26S proteasome. However, the precise nature of the complex is unknown. We used pollen extracts of a transgenic plant over-expressing GFP-fused S₂-SLF1 (SLF1 of S ₂-haplotype) for co-immunoprecipitation (Co-IP) followed by mass spectrometry (MS). We identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (an Rbx1). To validate the results, we raised transgenic plants over-expressing PiSSK1:FLAG:GFP and used pollen extracts for Co-IP–MS. The results confirmed the presence of PiCUL1-P and PiRBX1 in the complex and identified two different SLFs as the F-box protein component. Thus, all but Rbx1 of the complex may have evolved in SI, and all SLFs may be the F-box component of similar complexes.     

Controllable Chain‐Length for Covalent Sulfur–Carbon Materials Enabling Stable and High‐Capacity Sodium Storage

Room temperature sodium–sulfur batteries have emerged as promising candidate for application in energy storage. However, the electrodes are usually obtained through infusing elemental sulfur into various carbon sources, and the precipitation of insoluble and irreversible sulfide species on the surface of carbon and sodium readily leads to continuous capacity degradation. Here, a novel strategy is demonstrated to prepare a covalent sulfur–carbon complex (SC‐BDSA) with high covalent‐sulfur concentration (40.1%) that relies on ? SO₃H (Benzenedisulfonic acid, BDSA) and SO₄^2? as the sulfur source rather than elemental sulfur. Most of the sulfur is exists in the form of O? S/C? S bridge‐bonds (short/long‐chain) whose features ensure sufficient interfacial contact and maintain high ionic/electronic conductivities of the sulfur–carbon cathode. Meanwhile, the carbon mesopores resulting from the thermal‐treated salt bath can confine a certain amount of sulfur and localize the diffluent polysulfides. Furthermore, the C? S_x? C bridges can be electrochemically broken at lower potential (<0.6 V vs Na/Na⁺) and then function as a capacity sponsor. And the R‐SO units can anchor the initially generated S_x^2? to form insoluble surface‐bound intermediates. Thus SC‐BDSA exhibits a specific capacity of 696 mAh g^?1 at 2500 mA g^?1 and excellent cycling stability for 1000 cycles with 0.035% capacity decay per cycle.     

Incest versus abstinence: reproductive trade‐offs between mate limitation and progeny fitness in a self‐incompatible invasive plant

Plant mating systems represent an evolutionary and ecological trade‐off between reproductive assurance through selfing and maximizing progeny fitness through outbreeding. However, many plants with sporophytic self‐incompatibility systems exhibit dominance interactions at the S‐locus that allow biparental inbreeding, thereby facilitating mating between individuals that share alleles at the S‐locus. We investigated this trade‐off by estimating mate availability and biparental inbreeding depression in wild radish from five different populations across Australia. We found dominance interactions among S‐alleles increased mate availability relative to estimates based on individuals that did not share S‐alleles. Twelve of the sixteen fitness variables were significantly reduced by inbreeding. For all the three life‐history phases evaluated, self‐fertilized offspring suffered a greater than 50% reduction in fitness, while full‐sib and half‐sib offspring suffered a less than 50% reduction in fitness. Theory indicates that fitness costs greater than 50% can result in an evolutionary trajectory toward a stable state of self‐incompatibility (SI). This study suggests that dominance interactions at the S‐locus provide a possible third stable state between SI and SC where biparental inbreeding increases mate availability with relatively minor fitness costs. This strategy allows weeds to establish in new environments while maintaining a functional SI system.