Hongsheng Zhou

Genomic characterization, phylogenetic comparison and differential expression of the cyclic nucleotide-gated channels gene family in pear (Pyrus bretchneideri Rehd.)

The cyclic nucleotide-gated channel (CNGC) family is involved in the uptake of various cations, such as Ca(2+), to regulate plant growth and respond to biotic and abiotic stresses. However, there is far less information about this family in woody plants such as pear. Here, we provided a genome-wide identification and analysis of the CNGC gene family in pear. Phylogenetic analysis showed that the 21 pear CNGC genes could be divided into five groups (I, II, III, IVA and IVB). The majority of gene duplications in pear appeared to have been caused by segmental duplication and occurred 32.94-39.14 million years ago. Evolutionary analysis showed that positive selection had driven the evolution of pear CNGCs. Motif analyses showed that Group I CNGCs generally contained 26 motifs, which was the greatest number of motifs in all CNGC groups. Among these, eight motifs were shared by each group, suggesting that these domains play a conservative role in CNGC activity. Tissue-specific expression analysis indicated that functional diversification of the duplicated CNGC genes was a major feature of long-term evolution. Our results also suggested that the P-S6 and PBC & hinge domains had co-evolved during the evolution. These results provide valuable information to increase our understanding of the function, evolution and expression analyses of the CNGC gene family in higher plants.

Apoplastic calmodulin promotes self-incompatibility pollen tube growth by enhancing calcium influx and reactive oxygen species concentration in Pyrus pyrifolia

Key messageThis study indicated that Ca2+, ROS and actin filaments were involved with CaM in regulating pollen tube growth and providing a potential way for overcoming pear self-incompatibility.AbstractCalmodulin (CaM) has been associated with various physiological and developmental processes in plants, including pollen tube growth. In this study, we showed that CaM regulated the pear pollen tube growth in a concentration-dependent bi-phasic response. Using a whole-cell patch-clamp configuration, we showed that apoplastic CaM induced a hyperpolarization-activated calcium ion (Ca2+) current, and anti-CaM largely inhibited this type of Ca2+ current. Moreover, upon anti-CaM treatment, the reactive oxygen species (ROS) concentration decreased and actin filaments depolymerized in the pollen tube. Interestingly, CaM could partially rescue the inhibition of self-incompatible pear pollen tube growth. This phenotype could be mediated by CaM-enhanced pollen plasma membrane Ca2+ current, tip-localized ROS concentration and stabilized actin filaments. These data indicated that Ca2+, ROS and actin filaments were involved with CaM in regulating pollen tube growth and provide a potential way for overcoming pear self-incompatibility.

Low temperature inhibits pollen tube growth by disruption of both tip-localized reactive oxygen species and endocytosis in Pyrus bretschneideri Rehd.

Low temperature (LT) negatively affects fertilization processes of flowering plants. Pollen tube growth is generally inhibited under LT stress; however, the mechanism(s) underlying this inhibition remain(s) largely unknown. Pollen tubes are tip-growing and the presence of tip-localized reactive oxygen species (ROS) is necessary for cellular functioning. Disruption of tip-localized ROS was observed in pear pollen tubes in vitro under low temperature of 4 °C (LT4). Diphenylene iodonium chloride, an NADPH oxidase (NOX) inhibitor, suppressed hydrogen peroxide formation in the cell walls of the subapical region in pear pollen tubes. Under LT4 stress, ROS disruption in pear pollen tubes mainly resulted from decreased NOX activity in the plasma membrane, indicating that NOX was the main source of ROS in this process. Moreover, LT4 remarkably decreased mitochondrial oxygen consumption and intracellular ATP production. The endocytosis, an energy-dependent process, disruption in pear pollen tubes under LT4 may be mediated by mitochondrial metabolic dysfunctions. Our data showed ROS and endocytosis events in pear pollen tubes responding to LT4 stress.

Gene-expression profile of developing pollen tube of Pyrus bretschneideri

Pollen is an ideal model system for investigation of cell growth. In order to better understand the molecular biology mechanisms of the process of pear pollen tube development, RNA sequencing (RNA-Seq) technology was used to characterize the expression of genes during four development stages of pear pollen, including mature pollen grains (MP), hydrated pollen grains (HP), growing pollen tubes (PT) and stopped-growth pollen tubes (SPT). The four libraries generated a total of 47,072,151 clean reads that were mapped and assembled into 21,394 genes. Transcripts from the four stages were classified into 38 functional subcategories. Between MP and HP, 305 genes were differentially expressed, and 502 genes were differentially expressed between HP and PT. More importantly, we have observed that 2208 genes were differentially expressed between PT and SPT, and this is the first report of the gene expression comparison between the two development stages. Eight of the differentially expressed genes were randomly selected to confirm the RNA-Seq results by quantitative real-time PCR (qRT-PCR). Taken together, this research provides a platform for future research on pear pollen tube growth and growth cessation.

Genome-wide identification and comparative analysis of the cation proton antiporters family in pear and four other Rosaceae species

The monovalent cation proton antiporters (CPAs) play essential roles in plant nutrition, development, and signal transduction by regulating ion and pH homeostasis of the cell. The CPAs of plants include the Na+/H+ exchanger, K+ efflux antiporter, and cation/H+ exchanger families. However, currently, little is known about the CPA genes in Rosaceae species. In this study, 220 CPA genes were identified from five Rosaceae species (Pyrus bretschneideri, Malus domestica, Prunus persica, Fragaria vesca, and Prunus mume), and 53 of which came from P. bretschneideri. Phylogenetic, structure, collinearity, and gene expression analyses were conducted on the entire CPA genes of pear. Gene expression data showed that 35 and 37 CPA genes were expressed in pear fruit and pollen tubes, respectively. The transcript analysis of some CPA genes under abiotic stress conditions revealed that CPAs may play an important role in pollen tubes growth. The results presented here will be useful in improving understanding of the complexity of the CPA gene family and will promote functional characterization in future studies.

Mitochondrial dysfunction mediated by cytoplasmic acidification results in pollen tube growth cessation in Pyrus pyrifolia

The length of pollen tubes grown in synthetic media is normally shorter than those grown in vivo. However, the mechanism(s) underlying the cessation of pollen tube growth under culture conditions remain(s) largely unknown. Here, we report a previously unknown correlation between vacuolar function and the cells ability to sustain mitochondrial functions in pear pollen tubes. The pear pollen tubes in vitro grew slowly after 15 hours post-cultured (HPC) and nearly ceased growth at 18 HPC. There was increased malondialdehyde content and membrane ion leakage at 15 HPC compared with 12 HPC. Furthermore, cytoplasmic acidification mainly mediated by decreased vacuolar H(+)-ATPase [V-ATPase, Enzyme Commission (EC) 3.6.1.3] activity was observed in pollen tubes after 15 HPC, and this further resulted in mitochondrial dysfunction, including mitochondrial structure disruption, mitochondrial membrane potential collapse and decreases in both oxygen consumption and ATP production. Our findings suggest that vacuoles and mitochondria intimately linked in regulating pollen tube elongation.

Evolutionary and Expression Analysis Provides Evidence for the Plant Glutamate-like Receptors Family is Involved in Woody Growth-related Function.

Glutamate-like receptors (GLRs) is a highly conserved family of ligand-gated ion channels, which have been associated with various physiological and developmental processes. Here, we investigated the evolutionary pattern of GLRs in plants. We observed that tandem duplications occupied the largest proportion of the plant GLR gene family expansion. Based on a phylogenetic tree, we suggested a new subfamily, GLR4, which is widespread in angiosperm but absence on Brassicales. Meanwhile, because GLR1 and GLR2 subfamilies were potential sister clades, we combined them into the GLR1&2 subfamily. A comparative analysis of plant GLR subfamilies revealed that selective forces shaped the GLR1&2 repertoires in the stems of eudicotyledons with distinct functional preferences. Moreover, GLR1&2 formed a species-specific highwoody-expanded subfamily, with preferential expression in the cambial-enriched and shoot apical meristem fractions of the highwood species. Together, these findings lay the foundation for evolutionary analysis of plant GLRs over the entire plant timescale and identified unique targets for manipulating the woody-growth behaviours of plant GLRs.

A method to isolate male gametic nuclei from pear pollen tubes

Summary The nucleus plays an essential role in regulating most biological activities in most living organisms. Pear (Pyrus bretshneideri) pollen is binucleate, possessing a vegetative nucleus and a generative nucleus. To reveal the nuclear proteome and to produce accurate physical maps of DNA sequences using fluorescence in situ hybridisation (FISH) analysis, it is essential to obtain high quality nuclei. Protocols to isolate intact nuclei usually involve liberating them from centrifuged cells, then filtering them using a nylon mesh. Releasing the nuclei is the most critical step to obtain high quality material from small amounts of tissue. Here, we describe an easy and efficient method to isolate and purify both the generative and vegetative nuclei from pear pollen tubes. The nuclei were liberated by grinding pollen tubes, filtered on a cell strainer with a pore size of 40 µm, then passing them through a second cell strainer with a mesh size of 25 µm. After these procedures, and purification through a 28% (v/v) PercollTM gradient by centrifugation, the isolated nuclei exhibited a high level of purity and integrity, with little debris, as confirmed using enzyme markers and 4’,6-diamidino-2-phenylindole (DAPI) staining. The purified nuclei were used to prepare nuclear proteins and DNA fibres. The present protocol proved suitable for the isolation of male gametic nuclei from in vitro-cultivated pear pollen tubes, for nuclear proteome analysis, and for the preparation of DNA fibres for FISH analysis.

PbGLR3.3 Regulates Pollen Tube Growth in the Mediation of Ca 2+ Influx in Pyrus bretschneideri

Glutamate receptors (GluRs) are sensors of extracellular signals; they play important roles in the regulation of multiple physiological and developmental processes in eukaryotes. However, their functional roles in fruit trees are largely unknown. Here, based on the pear genome database, which was established in this lab, we identified 34 PbGLRs in pear (Pyrus bretschneideri Rehd), and they were divided into four groups by phylogenetic analysis. In comparisons with other groups, phylogenetic analyses and structural information of the PbGLRs in group 3 suggest that these genes underlie specific characteristics. Among the ten genes in group 3, we observed that the expression of PbGLR3.3 increased gradually during pollen germination and continuous growth, indicating that this gene might play a vital role in the development of pear pollen tubes. Using a combination of antisense oligodeoxy nucleotides and Ca2+-sensitive fluorescent probe methods, we verified that PbGLR3.3 participates in DSer-elicited intracellular Ca2+ signaling and Ca2+ regulation of growth in pear pollen tubes.

A method for isolating mitochondria from pear pollen tubes

Summary In an integrated cell system, mitochondria play a pivotal role in energy metabolism and the maintenance of cellular homeostasis. In most cases, protocols for the purification of the mitochondrial fraction are based on blenderhomogenisation of the selected tissue and differential centrifugation of the cell homogenate in a Percoll™ gradient. The method of homogenisation usually represents the critical step which affects the yield and quality of the mitochondria isolated from plant cells. To isolate mitochondria from minute, mg-amounts of plant tissue (e.g., pollen tubes), we have developed a new method for the homogenisation of pollen tubes using a 30-µm pore size cell strainer. The isolated mitochondria were shown to be pure, intact, and functional, based on marker enzyme assays, transmission electron microscopy, and flow cytometry analysis, respectively. Furthermore, the isolated mitochondria were used to extract mitochondrial DNA and to analyse mitochondrial proteins. The new method was easy, reliable, and efficient.