The effect of temperature on pollen germination, pollen tube growth, and stigmatic receptivity in peach

Temperature is a major climatic factor that limits geographical distribution of plant species, and the reproductive phase has proven to be one of the most temperature-vulnerable stages. Here, we have used peach to evaluate the effect of temperature on some processes of the progamic phase, from pollination to the arrival of pollen tubes in the ovary. Within the range of temperatures studied, 20 degrees C in the laboratory and, on average, 5.7 degrees C in the field, the results show an accelerating effect of increasing temperature on pollen germination and pollen tube growth kinetics, as well as an increase in the number of pollen tubes that reach the style base. For the last two parameters, although the range of temperature registered in the field was much lower, the results obtained in the laboratory paralleled those obtained in the field. Increasing temperatures drastically reduced stigmatic receptivity. Reduction was sequential, with stigmas first losing the capacity to sustain pollen tube penetration to the transmitting tissue, then their capacity to offer support for pollen germination and, finally, their capacity to support pollen grain adhesion. Within a species-specific range of temperature, this apparent opposite effect of temperature on the male and female side could provide plants with the plasticity to withstand changing environmental effects, ensuring a good level of fertilization.     

NtGNL1 is involved in embryonic cell division patterning, root elongation, and pollen tube growth in tobacco

The function of the ARF-GEF family has drawn great attention recently, especially GNOM and GNL1, owing to their important role in plant development. A homolog of GBF was identified in Nicotiana tabacum, named NtGNL1, which is ubiquitously expressed throughout the tobacco life cycle. In NtGNL1 RNAi plants, irregular orientation of cell division and asynchronous cell development during early embryogenesis disrupted the symmetry of the developing embryo. In addition, root growth in transgenic lines was significantly slower than that in wild-type plants, although the structure of the root tip was largely intact. Pollen germination and pollen tube growth were also inhibited in the transgenic lines, and the tip of the pollen tube presented various aberrant morphologies in one of the transgenic lines. The phenotypes of different NtGNL1 RNAi transgenic lines suggest that the NtGNL1 is likely to be involved not only in embryogenesis and postembryonic development, but also in sexual reproduction; thus, NtGNL1 may play multiple and critical roles in plant development.     

Monomeric G‐actin is uniformly distributed in pollen tubes and is rapidly redistributed via cytoplasmic streaming during pollen tube growth

Dynamic assembly and disassembly of the actin cytoskeleton has been implicated in the regulation of pollen germination and subsequent tube growth. It is widely accepted that actin filaments are arrayed into distinct structures within different regions of the pollen tube. Maintenance of the equilibrium between monomeric globular actin (G‐actin) and filamentous actin (F‐actin) is crucial for actin assembly and array construction, and the local concentration of G‐actin thus directly impacts actin assembly. The localization and dynamics of G‐actin in the pollen tube, however, remain to be determined conclusively. To address this question, we created a series of fusion proteins between green fluorescent protein (GFP) and the Arabidopsis reproductive actin ACT11. Expression of a fusion protein with GFP inserted after methionine at position 49 within the DNase I‐binding loop of ACT11 (GFP_Met49–ACT11) rescued the phenotypes in act11 mutants. Consistent with the notion that the majority of actin is in its monomeric form, GFP_Met49–ACT11 and GFP fusion proteins of four other reproductive actins generated with the same strategy do not obviously label filamentous structures. In further support of the functionality of these fusion proteins, we found that they can be incorporated into filamentous structures in jasplakinolide (Jasp)‐treated pollen tubes. Careful observations showed that G‐actin is distributed uniformly in the pollen tube and is rapidly redistributed via cytoplasmic streaming during pollen tube growth. Our study suggests that G‐actin is readily available in the cytoplasm to support continuous actin polymerization during rapid pollen tube growth.     

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.     

LeSTIG1, an extracellular binding partner for the pollen receptor kinases LePRK1 and LePRK2, promotes pollen tube growth in vitro

As pollen tubes grow through the pistil they are thought to perceive and respond to diverse signals. The tomato pollen-specific receptor kinases LePRK1 and LePRK2 might participate in signaling during pollen tube growth. We previously showed that the extracellular domain of LePRK2 interacts with a pollen protein, LAT52, before but not after pollen germination. To determine whether LePRK2 might have different binding partner(s) after pollen germination, we characterized two more proteins that, like LAT52, were identified in yeast two-hybrid screens using the extracellular domains of LePRK1 and LePRK2 as baits. We show that LeSHY, a leucine-rich repeat protein from pollen, and LeSTIG1, a small cysteine-rich protein from pistil, can bind the extracellular domains of both LePRK1 and LePRK2 in vitro. In vitro binding assays with the extracellular domain of LePRK2 suggested that LeSTIG1 could displace binding of LAT52, consistent with the idea that LePRK1 and LePRK2 might interact with different ligands at different stages of pollen tube growth. Exogenous LeSTIG1 promotes pollen tube growth in vitro. The interaction of these pollen kinases with LeSTIG1 supports the notion that LePRK1 and LePRK2 are involved in mediating pollen-pistil interactions.     

Cotton LIM domain‐containing protein GhPLIM1 is specifically expressed in anthers and participates in modulating F‐actin

As one form of actin binding protein (ABP), LIM domain protein can trigger the formation of actin bundles during plant growth and development. In this study, a cDNA (designated GhPLIM1) encoding a LIM domain protein with 216 amino acid residues was identified from a cotton flower cDNA library. Quantitative RT‐PCR indicated that GhPLIM1 is specifically expressed in cotton anthers, and its expression levels are regulated during anther development of cotton. GhPLIM1:eGFP transformed cotton cells display a distributed network of eGFP fluorescence, suggesting that GhPLIM1 protein is mainly localised to the cell cytoskeleton. In vitro high‐speed co‐sedimentation and low co‐sedimentation assays indicate that GhPLIM1 protein not only directly binds actin filaments but also bundles F‐actin. Further biochemical experiments verified that GhPLIM1 protein can protect F‐actin against depolymerisation by Lat B. Thus, our data demonstrate that GhPLIM1 functions as an actin binding protein (ABP) in modulating actin filaments in vitro, suggesting that GhPLIM1 may be involved in regulating the actin cytoskeleton required for pollen development in cotton.     

Enzyme activities of Arabidopsis inositol polyphosphate kinases AtIPK2α and AtIPK2β are involved in pollen development,pollen tube guidance and embryogenesis

Inositol polyphosphate kinase (IPK2) is a key component of inositol polyphosphate signaling. There are two highly homologous inositol polyphosphate kinases (AtIPK2α and AtIPK2β) in Arabidopsis. Previous studies that overexpressed or reduced the expression of AtIPK2α and AtIPK2β revealed their roles in auxiliary shoot branching, abiotic stress responses and root growth. Here, we report that AtIPK2α and AtIPK2β act redundantly during pollen development, pollen tube guidance and embryogenesis. Single knock‐out mutants of atipk2α and atipk2β were indistinguishable from the wild type, whereas the atipk2α atipk2β double mutant could not be obtained. Detailed genetic and cytological investigations showed that the mutation of AtIPK2α and AtIPK2β resulted in severely reduced transmission of male gametophyte as a result of abnormal pollen development and defective pollen tube guidance. In addition, the early embryo development of the atipk2α atipk2β double mutant was also aborted. Expressing either catalytically inactive or substrate specificity‐altered variants of AtIPK2β could not rescue the male gametophyte and embryogenesis defects of the atipk2α atipk2β double mutant, implying that the kinase activity of AtIPK2 is required for pollen development, pollen tube guidance and embryogenesis. Taken together, our results provide genetic evidence for the requirement of inositol polyphosphate signaling in plant sexual reproduction.     

NbEXPA1, an α‐expansin,is plasmodesmata‐specific and a novel host factor for potyviral infection

Plasmodesmata (PD), unique to the plant kingdom, are structurally complex microchannels that cross the cell wall to establish symplastic communication between neighbouring cells. Viral intercellular movement occurs through PD. To better understand the involvement of PD in viral infection, we conducted a quantitative proteomic study on the PD‐enriched fraction from Nicotiana benthamiana leaves in response to infection by Turnip mosaic virus (TuMV). We report the identification of a total of 1070 PD protein candidates, of which 100 (≥2‐fold increase) and 48 (≥2‐fold reduction) are significantly differentially accumulated in the PD‐enriched fraction, when compared with protein levels in the corresponding healthy control. Among the differentially accumulated PD protein candidates, we show that an α‐expansin designated NbEXPA1, a cell wall loosening protein, is PD‐specific. TuMV infection downregulates NbEXPA1 mRNA expression and protein accumulation. We further demonstrate that NbEXPA1 is recruited to the viral replication complex via the interaction with NIb, the only RNA‐dependent RNA polymerase of TuMV. Silencing of NbEXPA1 inhibits plant growth and TuMV infection, whereas overexpression of NbEXPA1 promotes viral replication and intercellular movement. These data suggest that NbEXPA1 is a host factor for potyviral infection. This study not only generates a PD‐proteome dataset that is useful in future studies to expound PD biology and PD‐mediated virus–host interactions but also characterizes NbEXPA1 as the first PD‐specific cell wall loosening protein and its essential role in potyviral infection.     

The DC1‐domain protein VACUOLELESS GAMETOPHYTES is essential for development of female and male gametophytes in Arabidopsis

In this work we identified VACUOLELESS GAMETOPHYTES (VLG) as a DC1 domain‐containing protein present in the endomembrane system and essential for development of both female and male gametophytes. VLG was originally annotated as a gene coding for a protein of unknown function containing DC1 domains. DC1 domains are cysteine‐ and histidine‐rich zinc finger domains found exclusively in the plant kingdom that have been named on the basis of similarity with the C1 domain present in protein kinase C (PKC). In Arabidopsis, both male and female gametophytes are characterized by the formation of a large vacuole early in development; this is absent in vlg mutant plants. As a consequence, development is arrested in embryo sacs and pollen grains at the first mitotic division. VLG is specifically located in multivesicular bodies or pre‐vacuolar compartments, and our results suggest that vesicular fusion is affected in the mutants, disrupting vacuole formation. Supporting this idea, AtPVA12 – a member of the SNARE vesicle‐associated protein family and previously related to a sterol‐binding protein, was identified as a VLG interactor. A role for VLG is proposed mediating vesicular fusion in plants as part of the sterol trafficking machinery required for vacuole biogenesis in plants.     

The tomato I gene for Fusarium wilt resistance encodes an atypical leucine‐rich repeat receptor‐like protein whose function is nevertheless dependent on SOBIR1 and SERK3/BAK1

We have identified the tomato I gene for resistance to the Fusarium wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol) and show that it encodes a membrane‐anchored leucine‐rich repeat receptor‐like protein (LRR‐RLP). Unlike most other LRR‐RLP genes involved in plant defence, the I gene is not a member of a gene cluster and contains introns in its coding sequence. The I gene encodes a loopout domain larger than those in most other LRR‐RLPs, with a distinct composition rich in serine and threonine residues. The I protein also lacks a basic cytosolic domain. Instead, this domain is rich in aromatic residues that could form a second transmembrane domain. The I protein recognises the Fol Avr1 effector protein, but, unlike many other LRR‐RLPs, recognition specificity is determined in the C‐terminal half of the protein by polymorphic amino acid residues in the LRRs just preceding the loopout domain and in the loopout domain itself. Despite these differences, we show that I/Avr1‐dependent necrosis in Nicotiana benthamiana depends on the LRR receptor‐like kinases (RLKs) SERK3/BAK1 and SOBIR1. Sequence comparisons revealed that the I protein and other LRR‐RLPs involved in plant defence all carry residues in their last LRR and C‐terminal LRR capping domain that are conserved with SERK3/BAK1‐interacting residues in the same relative positions in the LRR‐RLKs BRI1 and PSKR1. Tyrosine mutations of two of these conserved residues, Q922 and T925, abolished I/Avr1‐dependent necrosis in N. benthamiana, consistent with similar mutations in BRI1 and PSKR1 preventing their interaction with SERK3/BAK1.     

The DUF1996 and WSC domain‐containing protein Wsc1I acts as a novel sensor of multiple stress cues in Beauveria bassiana

Wsc1I homologues featuring both an N‐terminal DUF1996 (domain of unknown function 1996) and a C‐terminal WSC (cell wall stress‐responsive component) domain exist in filamentous fungi but have never been functionally characterized. Here, Wsc1I is shown to localize in the vacuoles and cell wall/membrane of the insect mycopathogen Beauveria bassiana and hence linked to cell membrane‐ and vacuole‐related cellular events. In B. bassiana, deletion of Wsc1I resulted in marked increases of hyphal and conidial sensitivities to hyperosmotic agents, oxidants, cell wall perturbing chemicals, and metal cations (Cu²⁺, Zn²⁺, Fe²⁺, and Mg²⁺) despite slight impact on normal growth and conidiation. Conidia produced by the deletion mutant showed not only reduced tolerance to both 45°C heat and UVB irradiation but also attenuated virulence to a susceptible insect through normal cuticle infection or cuticle‐bypassing infection. Importantly, phosphorylation of the mitogen‐activated protein kinase Hog1 was largely attenuated or nearly abolished in the Wsc1I‐free cells triggered with hyperosmotic, oxidative, or cell wall perturbing stress. All changes were well restored by targeted gene complementation. Our findings highlight a novel role of Wsc1I in sensing multiple stress cues upstream of the Hog1 signalling pathway and its pleiotropic effects in B. bassiana.     

Inhibition of bromodomain‐containing protein 4 ameliorates oxidative stress–mediated apoptosis and cartilage matrix degeneration through activation of NF‐E2–related factor 2‐heme oxygenase‐1 signaling in rat chondrocytes

During the progression of osteoarthritis, dysregulation of extracellular matrix (ECM) anabolism, abnormal generation of reactive oxygen species, and proteolytic enzymes have been shown to accelerate the degradation process of cartilage. The purpose of the current study was to investigate the functional role of bromodomain‐containing protein 4 (BRD4) in hydrogen peroxide (H₂O₂)–stimulated chondrocyte injury and delineate the underlying molecular mechanisms. We observed that the expression BRD4 was markedly elevated in rat chondrocytes after H₂O₂ stimulation. Additionally, inhibition of BRD4 using small interfering RNA or JQ1 (a selective potent chemical inhibitor) led to repression of H₂O₂‐induced oxidative stress, as revealed by a decrease in the reactive oxygen species production accompanied by a decreased malondialdehyde content, along with increased activities of antioxidant markers superoxide dismutase, catalase, and glutathione peroxidase on exposure of chondrocytes to H₂O₂. Meanwhile, depletion of BRD4 led to repress the oxidative stress–induced apoptosis of chondrocytes triggered by H₂O₂ accompanied by an increase in the expression of anti‐apoptotic Bcl‐2 and a decrease in the expression of pro‐apoptotic Bax and caspase 3 as well as attenuated caspase 3 activity. Moreover, knockdown of BRD4 or treatment with JQ1 markedly attenuated ECM deposition, reflected in a marked upregulation of proteoglycans collagen type II and aggrecan as well as downregulation of ECM–degrading enzymes matrix metalloproteinase 13 and A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS‐5). More importantly, inhibition of BRD4‐activated NF‐E2–related factor 2 (Nrf2)–heme oxygenase‐1 signaling. Mechanistically, the protective effect of BRD4 inhibition on H₂O₂‐stimulated apoptosis and cartilage matrix degeneration was markedly abrogated by Nrf2 depletion. Altogether, we concluded that the protective effect of BRD4 inhibition against oxidative stress–mediated apoptosis and cartilage matrix degeneration occurred through Nrf2–heme oxygenase‐1 signaling, implying that BRD4 inhibition may be a more effective therapeutic strategy against osteoarthritis.     

ITN1, a novel gene encoding an ankyrin-repeat protein that affects the ABA-mediated production of reactive oxygen species and is involved in salt-stress tolerance in Arabidopsis thaliana

Salt stress and abscisic acid (ABA) induce accumulation of reactive oxygen species (ROS) in plant cells. ROS not only act as second messengers for the activation of salt-stress responses, but also have deleterious effects on plant growth due to their cytotoxicity. Therefore, the timing and degree of activation of ROS-producing or ROS-scavenging enzymes must be tightly regulated under salt-stress conditions. We identified a novel locus of Arabidopsis, designated itn1 (increased tolerance to NaCl1), whose disruption leads to increased salt-stress tolerance in vegetative tissues. ITN1 encodes a transmembrane protein with an ankyrin-repeat motif that has been implicated in diverse cellular processes such as signal transduction. Comparative microarray analysis between wild-type and the itn1 mutant revealed that induction of genes encoding the ROS-producing NADPH oxidases (RBOHC and RBOHD) under salt-stress conditions was suppressed in the mutant. This suppression was accompanied by a corresponding reduction in ROS accumulation. The ABA-induced expression of RBOHC and RBOHD was also suppressed in the mutant, as was the case for RD29A, an ABA-inducible marker gene. However, the ABA-induced expression of another marker gene, RD22, was not impaired in the mutant. These results suggest that the itn1 mutation partially impairs ABA signaling pathways, possibly leading to the reduction in ROS accumulation under salt-stress conditions. We discuss the possible mechanisms underlying the salt-tolerant phenotype of the itn1 mutant.     

Tumour necrosis factor‐α‐induced protein 8‐like 2 is a novel regulator of proliferation,migration, and invasion in human rectal adenocarcinoma cells

Tumour necrosis factor‐α‐induced protein 8‐like 2 (TIPE2) is a tumour suppressor in many types of cancer. However, the mechanism of action of TIPE2 on the growth of rectal adenocarcinoma is unknown. Our results showed that the expression levels of TIPE2 in human rectal adenocarcinoma tissues were higher than those in adjacent non‐tumour tissues. Overexpression of TIPE2 reduced the proliferation, migration, and invasion of human rectal adenocarcinoma cells and down‐regulation of TIPE2 showed reverse effects. TIPE2 overexpression increased apoptosis through down‐regulating the expression levels of Wnt3a, phospho (p)‐β‐Catenin, and p‐glycogen synthase kinase‐3β in rectal adenocarcinoma cells, however, TIPE2 knockdown exhibited reverse trends. TIPE2 overexpression decreased autophagy by reducing the expression levels of p‐Smad2, p‐Smad3, and transforming growth factor‐beta (TGF‐β) in rectal adenocarcinoma cells, however, TIPE2 knockdown showed opposite effects. Furthermore, TIPE2 overexpression reduced the growth of xenografted human rectal adenocarcinoma, whereas TIPE2 knockdown promoted the growth of rectal adenocarcinoma tumours by modulating angiogenesis. In conclusion, TIPE2 could regulate the proliferation, migration, and invasion of human rectal adenocarcinoma cells through Wnt/β‐Catenin and TGF‐β/Smad2/3 signalling pathways. TIPE2 is a potential therapeutic target for the treatment of rectal adenocarcinoma.