Xue-Qin Zhang

VANGUARD1 Encodes a Pectin Methylesterase That Enhances Pollen Tube Growth in the Arabidopsis Style and Transmitting Tract

In flowering plants, penetration of the pollen tube through stigma, style, and transmitting tract is essential for delivery of sperm nuclei to the egg cells embedded deeply within female tissues. Despite its importance in plant reproduction, little is known about the underlying molecular mechanisms that regulate the navigation of the pollen tube through the stigma, style, and transmitting tract. Here, we report the identification and characterization of an Arabidopsis thaliana gene, VANGUARD1 (VGD1) that encodes a pectin methylesterase (PME)-homologous protein of 595 amino acids and is required for enhancing the growth of pollen tubes in the style and transmitting tract tissues. VGD1 was expressed specifically in pollen grain and the pollen tube. The VGD1 protein was distributed throughout the pollen grain and pollen tube, including the plasma membrane and cell wall. Functional interruption of VGD1 reduced PME activity in the pollen to 82% of the wild type and greatly retarded the growth of the pollen tube in the style and transmitting tract, resulting in a significant reduction of male fertility. In addition, the vgd1 pollen tubes were unstable and burst more frequently when germinated and grown on in vitro culture medium, compared with wild-type pollen tubes. Our study suggests that the VGD1 product is required for growth of the pollen tube, possibly via modifying the cell wall and enhancing the interaction of the pollen tube with the female style and transmitting tract tissues.

Overexpression of TAPETUM DETERMINANT1 Alters the Cell Fates in the Arabidopsis Carpel and Tapetum via Genetic Interaction with EXCESS MICROSPOROCYTES1/EXTRA SPOROGENOUS CELLS

Previously, we reported that the TAPETUM DETERMINANT1 (TPD1) gene is required for specialization of tapetal cells in the Arabidopsis (Arabidopsis thaliana) anther. The tpd1 mutant is phenotypically identical to the excess microsporocytes1 (ems1)/extra sporogenous cells (exs) mutant. The TPD1 and EMS1/EXS genes may function in the same developmental pathway in the Arabidopsis anther. Here, we further report that overexpression of TPD1 alters the cell fates in the Arabidopsis carpel and tapetum. When TPD1 was expressed ectopically in the wild-type Arabidopsis carpel, the number of cells in the carpel increased significantly, showing that the ectopic expression of TPD1 protein could activate the cell division in the carpel. Furthermore, the genetic analysis showed that the activation of cell division in the transgenic carpel by TPD1 was dependent on EMS1/EXS, as it did not happen in the ems1/exs mutant. This result further suggests that TPD1 regulates cell fates in coordination with EMS1/EXS. Moreover, overexpression of TPD1 in tapetal cells also delayed the degeneration of tapetum. The TPD1 may function not only in the specialization of tapetal cells but also in the maintenance of tapetal cell fate.

A mutation in Thermosensitive Male Sterile 1, encoding a heat shock protein with DnaJ and PDI domains, leads to thermosensitive gametophytic male sterility in Arabidopsis.

In most flowering plant species, pollination and fertilization occur during the hot summer, so plants must have evolved a mechanism that ensures normal growth of their pollen tubes at high temperatures. Despite its importance to plant reproduction, little is known about the molecular basis of thermotolerance in pollen tubes. Here we report the identification and characterization of a novel Arabidopsis gene, Thermosensitive Male Sterile 1 (TMS1), which plays an important role in thermotolerance of pollen tubes. TMS1 encodes a Hsp40-homologous protein with a DnaJ domain and an a_ERdj5_C domain found in protein disulfide isomerases (PDI). Purified TMS1 expressed in Escherichia coli (BL21 DE3) had the reductive activity of PDI. TMS1 was expressed in pollen grains, pollen tubes and other vegetative tissues, including leaves, stems and roots. Heat shock treatment at 37 degrees C increased its expression levels in growing pollen tubes as well as in vegetative tissues. A knockout mutation in TMS1 grown at 30 degrees C had greatly retarded pollen tube growth in the transmitting tract, resulting in a significant reduction in male fertility. Our study suggests that TMS1 is required for thermotolerance of pollen tubes in Arabidopsis, possibly by functioning as a co-molecular chaperone.

MYB97, MYB101 and MYB120 Function as Male Factors That Control Pollen Tube-Synergid Interaction in Arabidopsis thaliana Fertilization

Pollen tube reception involves a pollen tube-synergid interaction that controls the discharge of sperm cells into the embryo sac during plant fertilization. Despite its importance in the sexual reproduction of plants, little is known about the role of gene regulation in this process. We report here that the pollen-expressed transcription factors MYB97, MYB101 and MYB120 probably control genes whose encoded proteins play important roles in Arabidopsis thaliana pollen tube reception. They share a high amino acid sequence identity and are expressed mainly in mature pollen grains and pollen tubes. None of the single or double mutants of these three genes exhibited any visible defective phenotype. Although the myb97 myb101 myb120 triple mutant was not defective in pollen development, pollen germination, pollen tube growth or tube guidance, the pollen tubes of the triple mutants exhibited uncontrolled growth and failed to discharge their sperm cells after entering the embryo sac. In addition, the myb97 myb101 myb120 triple mutation significantly affected the expression of a group of pollen-expressed genes in mature pollen grains. All these results indicate that MYB97, MYB101 and MYB120 participate in pollen tube reception, possibly by controlling the expression of downstream genes.

Mutations in the Arabidopsis Nuclear-Encoded Mitochondrial Phage-Type RNA Polymerase Gene RPOTm Led to Defects in Pollen Tube Growth, Female Gametogenesis and Embryogenesis

The mitochondrial genes in Arabidopsis thaliana are transcribed by a small family of nuclear-encoded T3/T7 phage-type RNA polymerases (RPOTs). At least two nuclear-encoded RPOTs (RPOTm and RPOTmp) are located in mitochondria in A. thaliana. Their genetic roles are largely unknown. Here we report the characterization of novel mutations in the A. thaliana RPOTm gene. The mutations did not affect pollen formation, but significantly retarded the growth of the rpoTm mutant pollen tubes and had an impact on the fusion of the polar nuclei in the rpoTm mutant embryo sacs. Moreover, development of the rpoTm/- mutant embryo was arrested at the globular stage. The rpoTm rpoTmp double mutation could enhance the rpoTm mutant phenotype. Expression of RPOTmp under control of the RPOTm promoter could not complement the phenotype of the rpoTm mutations. All these data indicate that RPOTm is important for normal pollen tube growth, female gametogenesis and embryo development, and has distinct genetic and molecular roles in plant development, which cannot be replaced by RPOTmp.

MALE GAMETOPHYTE DEFECTIVE 2, Encoding a Sialyltransferase-like Protein, is Required for Normal Pollen Germination and Pollen Tube Growth in Arabidopsis

Sialyltransferases (SiaTs) exist widely in vertebrates and play important roles in a variety of biological processes. In plants, several genes have also been identified to encode the proteins that share homology with the vertebrate SiaTs. However, very little is known about their functions in plants. Here we report the identification and characterization of a novel Arabidopsis gene, MALE GAMETOPHYTE DEFECTIVE 2 (MGP2) that encodes a sialyltransferase-like protein. MGP2 was expressed in all tissues including pollen grains and pollen tubes. The MGP2 protein was targeted to Golgi apparatus. Knockout of MGP2 significantly inhibited the pollen germination and retarded pollen tube growth in vitro and in vivo, but did not affect female gametophytic functions. These results suggest that the sialyltransferase-like protein MGP2 is important for normal pollen germination and pollen tube growth, giving a novel insight into the biological roles of the sialyltransferase-like proteins in plants.

GNOM-LIKE 2, encoding an adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factor protein homologous to GNOM and GNL1, is essential for pollen germination in Arabidopsis.

In flowering plants, male gametes are delivered to female gametophytes by pollen tubes. Although it is important for sexual plant reproduction, little is known about the genetic mechanism that controls pollen germination and pollen tube growth. Here we report the identification and characterization of two novel mutants, gnom-like 2-1 (gnl2-1) and gnl2-2 in Arabidopsis thaliana, in which the pollen grains failed to germinate in vitro and in vivo. GNL2 encodes a protein homologous to the adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factors, GNOM and GNL1 that are involved in endosomal recycling and endoplasmic reticulum-Golgi vesicular trafficking. It was prolifically expressed in pollen grains and pollen tubes. The results of the present study suggest that GNL2 plays an important role in pollen germination.

MALE GAMETOPHYTE DEFECTIVE 1, Encoding the FAd Subunit of Mitochondrial F1F0-ATP Synthase, is Essential for Pollen Formation in Arabidopsis thaliana

In flowering plants, pollen formation is a complex process and strictly controlled by genetic factors. Although thousands of genes have been identified to be highly or specifically expressed in pollen grains, little is known about the functions and regulatory mechanisms of the genes in pollen formation. Here we report the characterization of a novel gene, MALE GAMETOPHYTE DEFECTIVE 1 (MGP1), that is essential for pollen formation in Arabidopsis thaliana. MGP1 encodes the F(A)d subunit of mitochondrial F(1)F(0)-ATP synthase in Arabidopsis. It was highly expressed in pollen grains at the later developmental stage. Mutation in MGP1 led to destruction of the mitochondria in pollen grains at the dehydration stage and subsequently death of the pollen grains. These results suggested that MGP1 plays important roles in pollen formation, possibly by regulating the activity of mitochondrial F(1)F(0)-ATP synthase in Arabidopsis pollen grains.

MARIS plays important roles in Arabidopsis pollen tube and root hair growth

In flowering plants, male gametes are delivered to female gametes for double fertilization through pollen tubes. Therefore, pollen tube growth is crucial for double fertilization. Despite its importance to sexual reproduction, genetic mechanisms of pollen tube growth remain poorly understood. In this study, we characterized the receptor-like cytoplasmic protein kinase (RLCK) gene, MARIS (MRI) that plays critical roles in pollen tube growth. MRI is preferentially expressed in pollen grains, pollen tubes and roots. Mutation in MRI by a Ds insertion led to a burst of pollen tubes after pollen germination. Pollen-rescue assay by pollen and pollen tube-specific expression of MRI in the mri-4 mutant showed that loss of MRI function also severely affected root hair elongation. MRI protein interacted with the protein kinase OXIDATIVE SIGNAL INDUCIBLE1 (OXI1) in the in vitro and in vivo assays, which functions in plant defence and root hair development, and was phosphorylated by OXI1 in vitro. Our results suggest that MRI plays important roles in pollen tube growth and may function in root hair elongation through interaction with OXI1.

Pollen-Expressed Transcription Factor 2 Encodes a Novel Plant-Specific TFIIB-Related Protein that Is Required for Pollen Germination and Embryogenesis in Arabidopsis

Pollen germination and embryogenesis are important to sexual plant reproduction. The processes require a large number of genes to be expressed. Transcription of eukaryotic nuclear genes is accomplished by three conserved RNA polymerases acting in association with a set of auxiliary general transcription factors (GTFs), including B-type GTFs. The roles of B-type GTFs in plant reproduction remain poorly understood. Here we report functional characterization of a novel plant-specific TFIIB-related gene PTF2 in Arabidopsis. Mutation in PTF2 caused failure of pollen germination. Pollen-rescue revealed that the mutation also disrupted embryogenesis and resulted in seed abortion. PTF2 is expressed prolifically in developing pollen and the other tissues with active cell division and differentiation, including embryo and shoot apical meristem. The PTF2 protein shares a lower amino acid sequence similarity with other known TFIIB and TFIIB-related proteins in Arabidopsis. It can interact with TATA-box binding protein 2 (TBP2) and bind to the double-stranded DNA (dsDNA) as the other known TFIIB and TFIIB-related proteins do. In addition, PTF2 can form a homodimer and interact with the subunits of RNA polymerases (RNAPs), implying that it may be involved in the RNAPs transcription. These results suggest that PTF2 plays crucial roles in pollen germination and embryogenesis in Arabidopsis, possibly by regulating gene expression through interaction with TBP2 and the subunits of RNAPs.