Molecular Plant - Advance Access

Transcriptome-Based Examination of Putative Pollen Allergens of Rice (Oryza sativa ssp. japonica)

Russell, S. D., Bhalla, P. L., Singh, M. B. — 2008-07-21

Pollen allergens are among the most abundantly transcribed and translated products in the life history of plants, and particularly grasses. To identify different pollen allergens in rice, putative allergens were identified in the rice genome and their expression characterized using the Affymetrix 57K rice GeneChip microarray. Among the most abundant pollen-specific candidate transcripts were Ory s 1 beta-expansin, Ory s 2, Ory s 7 EF hand, Ory s 11, Ory s 12 profilin A, Ory s 23, glycosyl hydrolase family 28 (polygalacturonase), and FAD binding proteins. Highly expressed pollen proteins are frequently present in multiple copy numbers, sometimes with mirror images located on nearby regions of the opposite DNA strand. Many of these are intronless and inserted as copies that retain nearly exact copies of their regulatory elements. Ory s 23 reflects low variability and high copy number, suggesting recent gene amplification. Some copies contain pseudogenes, which may reflect their origin through activity of retrotransposition; some putative allergenic sequences bear fusion products with repeat sequences of transposable elements (LTRs). The abundance of nearby repetitive sequences, activation of transposable elements, and high production of mRNA transcripts appear to coincide in pollen and may contribute to a syndrome in which highly transcribed proteins may be copied and inserted with streamlined features for translation, including grouping and removal of introns.

A Novel C2-Domain Phospholipid-Binding Protein, OsPBP1, Is Required for Pollen Fertility in Rice

Yang, W.-Q., Lai, Y., Li, M.-N., Xu, W.-Y., Xue, Y.-B. — 2008-07-21

Pollen fertility is a crucial factor for successful pollination and essential for seed formation. Recent studies have suggested that a diverse range of internal and external factors, signaling components and their related pathways are likely involved in pollen fertility. Here, we report a single C2-domain containing protein, OsPBP1, initially identified through cDNA microarray analysis. OsPBP1 is a single copy gene and preferentially expressed in pistil and pollen but down-regulated by pollination. OsPBP1 had a calcium concentration-dependent phospholipid-binding activity and was localized mainly in cytoplasm and nucleus, but translocated onto the plasma membrane in response to an intracellular Ca²⁺ increase. Pollen grains of antisense OsPBP1 transgenic lines were largely nonviable, germinated poorly in vitro and of low fertility. OsPBP1 protein was localized in a region peripheral to pollen wall and vesicles of elongating pollen tube, and its repressed expression reduced substantially this association and led to alteration of microfilament polymerization during pollen germination. Taken together, these results indicate that OsPBP1 is a novel functional C2-domain phospholipids-binding protein that is required for pollen fertility likely by regulating Ca²⁺ and phospholipid signaling pathways.

Targeting of Pollen Tubes to Ovules Is Dependent on Nitric Oxide (NO) Signaling

Prado, A. M., Colaco, R., Moreno, N., Silva, A. C., Feijo, J. A. — 2008-06-27

The guidance signals that drive pollen tube navigation inside the pistil and micropyle targeting are still, to a great extent, unknown. Previous studies in vitro showed that nitric oxide (NO) works as a negative chemotropic cue for pollen tube growth in lily (Lilium longiflorum). Furthermore, Arabidopsis thaliana Atnos1 mutant plants, which show defective NO production, have reduced fertility. Here, we focus in the role of NO in the process of pollen–pistil communication, using Arabidopsis in-vivo and lily semi-vivo assays. Cross-pollination between wild-type and Atnos1 plants shows that the mutation affects the pistil tissues in a way that is compatible with abnormal pollen tube guidance. Moreover, DAF-2DA staining for NO in kanadi floral mutants showed the presence of NO in an asymmetric restricted area around the micropyle. The pollen–pistil interaction transcriptome indicates a time-course-specific modulation of transcripts of AtNOS1 and two Nitrate Reductases (nr1 and nr2), which collectively are thought to trigger a putative NO signaling pathway. Semi-vivo assays with isolated ovules and lily pollen further showed that NO is necessary for micropyle targeting to occur. This evidence is supported by CPTIO treatment with subsequent formation of balloon tips in pollen tubes facing ovules. Activation of calcium influx in pollen tubes partially rescued normal pollen tube morphology, suggesting that this pathway is also dependent on Ca²⁺ signaling. A role of NO in modulating Ca²⁺ signaling was further substantiated by direct imaging the cytosolic free Ca²⁺ concentration during NO-induced re-orientation, where two peaks of Ca²⁺ occur—one during the slowdown/stop response, the second during re-orientation and growth resumption. Taken together, these results provide evidence for the participation of NO signaling events during pollen–pistil interaction. Of special relevance, NO seems to directly affect the targeting of pollen tubes to the ovule's micropyle by modulating the action of its diffusible factors.

A Mitochondrial Magnesium Transporter Functions in Arabidopsis Pollen Development

2008-06-27

Magnesium is an abundant divalent cation in plant cells and plays a critical role in many physiological processes. We have previously described the identification of a 10-member Arabidopsis gene family encoding putative magnesium transport (MGT) proteins. Here, we report that a member of the MGT family, AtMGT5, functions as a dual-functional Mg-transporter that operates in a concentration-dependent manner, namely it serves as a Mg-importer at micromolar levels and facilitates the efflux in the millimolar range. The AtMGT5 protein is localized in the mitochondria, suggesting that AtMGT5 mediates Mg-trafficking between the cytosol and mitochondria. The AtMGT5 gene was exclusively expressed in anthers at early stages of flower development. Examination of two independent T-DNA insertional mutants of AtMGT5 gene demonstrated that AtMGT5 played an essential role for pollen development and male fertility. This study suggests a critical role for Mg²⁺ transport between cytosol and mitochondria in male gametogenesis in plants.

Biochemical Models for S-RNase-Based Self-Incompatibility

Hua, Z.-H., Fields, A., Kao, T.-h. — 2008-06-26

S-RNase-based self-incompatibility (SI) is a genetically determined self/non-self-recognition process employed by many flowering plant species to prevent inbreeding and promote outcrosses. For the Plantaginaceae, Rosaceae and Solanaceae, it is now known that S-RNase and S-locus F-box (two multiple allelic genes at the S-locus) determine the female and male specificity, respectively, during SI interactions. However, how allelic products of these two genes interact inside pollen tubes to result in specific growth inhibition of self-pollen tubes remains to be investigated. Here, we review all the previously proposed biochemical models and discuss whether their predictions are consistent with all SI phenomena, including competitive interaction where SI breaks down in pollen that carries two different pollen S-alleles. We also discuss these models in light of the recent findings of compartmentalization of S-RNases in both incompatible and compatible pollen tubes. Lastly, we summarize the results from our recent biochemical studies of PiSLF (Petunia inflata SLF) and S-RNase, and present a new model for the biochemical mechanism of SI in the Solanaceae. The tenet of this model is that a PiSLF preferentially interacts with its non-self S-RNases in the cytoplasm of a pollen tube to result in the assembly of an E3-like complex, which then mediates ubiquitination and degradation of non-self S-RNases through the ubiquitin–26S proteasome pathway. This model can explain all SI phenomena and, at the same time, has raised new questions for further study.

SWI1 Is Required for Meiotic Chromosome Remodeling Events

Boateng, K. A., Yang, X., Dong, F., Owen, H. A., Makaroff, C. A. — 2008-06-26

The Arabidopsis dsy10 mutant was previously identified as being defective in the synapsis of meiotic chromosomes resulting in male and female sterility. We report here the molecular analysis of the mutation and show that it represents a T-DNA insertion in the third exon of the SWI1 gene. Four mutations have now been identified in SWI1, several of which exhibit different phenotypes. For example, the swi1-1 and dyad mutations only affect meiosis in megasporocytes, while the swi1-2 and dsy10 mutations block both male and female meiosis. Furthermore, as part of a detailed cytological characterization of dsy10 meiocytes, we identified several differences during male meiosis between the swi1-2 and dys10 mutants, including variations in the formation of axial elements, the distribution of cohesin proteins and the timing of the premature loss of sister chromatid cohesion. We demonstrate that dsy10 represents a complete loss-of-function mutation, while a truncated form of SWI1 is expressed during meiosis in swi1-2 plants. We further show that dys10 meiocytes exhibit alterations in modified histone patterns, including acetylated histone H3 and dimethylated histone H3-Lysine 4.

Microscopy and Bioinformatic Analyses of Lipid Metabolism Implicate a Sporophytic Signaling Network Supporting Pollen Development in Arabidopsis

Wang, Y., Wu, H., Yang, M. — 2008-06-25

The Arabidopsis sporophytic tapetum undergoes a programmed degeneration process to secrete lipid and other materials to support pollen development. However, the molecular mechanism regulating the degeneration process is unknown. To gain insight into this molecular mechanism, we first determined that the most critical period for tapetal secretion to support pollen development is from the vacuolate microspore stage to the early binucleate pollen stage. We then analyzed the expression of enzymes responsible for lipid biosynthesis and degradation with available in-silico data. The genes for these enzymes that are expressed in the stamen but not in the concurrent uninucleate microspore and binucleate pollen are of particular interest, as they presumably hold the clues to unique molecular processes in the sporophytic tissues compared to the gametophytic tissue. No gene for lipid biosynthesis but a single gene encoding a patatin-like protein likely for lipid mobilization was identified based on the selection criterion. A search for genes co-expressed with this gene identified additional genes encoding typical signal transduction components such as a leucine-rich repeat receptor kinase, an extra-large G-protein, other protein kinases, and transcription factors. In addition, proteases, cell wall degradation enzymes, and other proteins were also identified. These proteins thus may be components of a signaling network leading to degradation of a broad range of cellular components. Since a broad range of degradation activities is expected to occur only in the tapetal degeneration process at this stage in the stamen, it is further hypothesized that the signaling network acts in the tapetal degeneration process.

Tapetum Degeneration Retardation is Critical for Aliphatic Metabolism and Gene Regulation during Rice Pollen Development

2008-06-23

As a complex wall system in flowering plants, the pollen outer wall mainly contains aliphatic sporopollenin; however, the mechanism for synthesizing these lipidic precursors during pollen development remains less well understood. Here, we report on the function of the rice tapetum-expressing TDR (Tapetum Degeneration Retardation) gene in aliphatic metabolism and its regulatory role during rice pollen development. The observations of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses suggested that pollen wall formation was significantly altered in the tdr mutant. The contents of aliphatic compositions of anther were greatly changed in the tdr mutant revealed by GC–MS (gas chromatography–mass spectrometry) testing, particularly less accumulated in fatty acids, primary alcohols, alkanes and alkenes, and an abnormal increase in secondary alcohols with carbon lengths from C29 to C35 in tdr. Microarray data revealed that a group of genes putatively involved in lipid transport and metabolism were significantly altered in the tdr mutant, indicating the critical role of TDR in the formation of the pollen wall. Also, a wide range of genes (236 in total—154 up-regulated and 82 down-regulated) exhibited statistically significant expressional differences between wild-type and tdr. In addition to its function in promoting tapetum PCD, TDR possibly plays crucial regulatory roles in several basic biological processes during rice pollen development.

New microsome-associated HT-family proteins from Nicotiana respond to pollination and define an HT/NOD-24 protein family

Kondo, K., McClure, B. — 2008-06-23

HT-family proteins have been identified in Nicotiana, Solanum, and Petunia. HT-B-type proteins are implicated in S-RNase-based self-incompatibility, but the functions of other family members are unknown. Screening for cDNA sequences with an expression pattern similar to HT-B in Nicotiana alata revealed a new group of small HT-family proteins, designated HT-M. HT-M proteins resemble HT-B in several respects: their pistil-specific expression pattern is indistinguishable from HT-B, they pellet with a microsome fraction, and their abundance decreases after pollination. Unlike HT-B, there is no S-specificity to this response, and RNAi experiments show that HT-M proteins are not necessary for self-incompatibility. Identification of a third group of pistil-specific HT-family proteins helps better define the characteristics of the family and allowed identification of putative new family members. By searching the databases with only the most conserved HT-family sequence elements, the signal sequence and cysteine motifs, we identified nodulin-24-like proteins and several small glycine-rich proteins as putative HT-family members. Like HT-M and HT-B, nodulin-24 is membrane associated. We propose that the conserved features in HT-family proteins are important for targeting or modification and refer to the broader family that includes both HT- and nodulin-24-like proteins as the HT/NOD-24-family.

The Dynamic Pollen Tube Cytoskeleton: Live Cell Studies Using Actin-Binding and Microtubule-Binding Reporter Proteins

2008-06-12

Pollen tubes elongate within the pistil to transport sperm cells to the embryo sac for fertilization. Growth occurs exclusively at the tube apex, rendering pollen tube elongation a most dramatic polar cell growth process. A hallmark pollen tube feature is its cytoskeleton, which comprises elaborately organized and dynamic actin microfilaments and microtubules. Pollen tube growth is dependent on the actin cytoskeleton; its organization and regulation have been examined extensively by various approaches, including fluorescent protein labeled actin-binding proteins in live cell studies. Using the previously described GFP-NtADF1 and GFP-LlADF1, and a new actin reporter protein NtPLIM2b-GFP, we re-affirm that the predominant actin structures in elongating tobacco and lily pollen tubes are long, streaming actin cables along the pollen tube shank, and a subapical structure comprising shorter actin cables. The subapical collection of actin microfilaments undergoes dynamic changes, giving rise to the appearance of structures that range from basket- or funnel-shaped, mesh-like to a subtle ring. NtPLIM2b-GFP is used in combination with a guanine nucleotide exchange factor for the Rho GTPases, AtROP-GEF1, to illustrate the use of these actin reporter proteins to explore the linkage between the polar cell growth process and its actin cytoskeleton. Contrary to the actin cytoskeleton, microtubules appear not to play a direct role in supporting the polar cell growth process in angiosperm pollen tubes. Using a microtubule reporter protein based on the microtubule end-binding protein from Arabidopsis AtEB1, GFP-AtEB1, we show that the extensive microtubule network in elongating pollen tubes displays varying degrees of dynamics. These reporter proteins provide versatile tools to explore the functional connection between major structural and signaling components of the polar pollen tube growth process.

Regulation of Arabidopsis Early Anther Development by the Mitogen-Activated Protein Kinases, MPK3 and MPK6, and the ERECTA and Related Receptor-Like Kinases

2008-06-03

Mitogen-activated protein kinase (MAPK) and leucine-rich repeat receptor-like kinase (LRR-RLK) signaling pathways have been shown to regulate diverse aspects of plant growth and development. In Arabidopsis, proper anther development relies on intercellular communication to coordinate cell proliferation and differentiation. Two closely related genes encoding MAPKs, MPK3 and MPK6, function redundantly in regulating stomatal patterning. Although the mpk6 mutant has reduced fertility, the function of MPK3 and MPK6 in anther development has not been characterized. Similarly, the ERECTA (ER), ERECTA-LIKE1 (ERL1) and ERL2 genes encoding LRR-RLKs function together to direct stomatal cell fate specification and the er-105 erl1-2 erl2-1 triple mutant is sterile. Because the mpk3 mpk6 double null mutant is embryo lethal, anther development was characterized in the viable mpk3/+ mpk6/– and er-105 erl1-2 erl2-1 mutants. We found that both mutant anthers usually fail to form one or more of the four anther lobes, with the er-105 erl1-2 erl2-1 triple mutant exhibiting more severe phenotypes than those of the mpk3/+ mpk6/– mutant. The somatic cell layers of the differentiated mutant lobes appeared larger and more disorganized than that of wild-type. In addition, the er-105 erl1-2 erl2-1 triple mutant has a reduced number of stamens, the majority of which possess completely undifferentiated or under-differentiated anthers. Furthermore, sometimes, the mpk3/+ mpk6/– mutant anthers do not dehisce, and the er-105 erl1-2 erl2-1 anthers were not observed to dehisce. Therefore, our results indicate that both ER/ERL1/ERL2 and MPK3/MPK6 play important roles in normal anther lobe formation and anther cell differentiation. The close functional relationship between these genes in other developmental processes and the similarities in anther developmental phenotypes of the two types of mutants reported here further suggest the possibility that these genes might also function in the same pathway to regulate anther cell division and differentiation.

GEX3, Expressed in the Male Gametophyte and in the Egg Cell of Arabidopsis thaliana, Is Essential for Micropylar Pollen Tube Guidance and Plays a Role during Early Embryogenesis

Alandete-Saez, M., Ron, M., McCormick, S. — 2008-05-25

Double fertilization in flowering plants occurs when the two sperm cells, carried by the pollen tube, are released in a synergid cell of the embryo sac and then fertilize the egg and the central cell. Proteins on the surfaces of the sperm, egg, central, and synergid cells might be important for guidance and recognition/fusion of the gametes. Here, we present functional analyses of Arabidopsis GEX3, which encodes a plasma membrane-localized protein that has homologs in other plants. GEX3 is expressed in both the vegetative and sperm cells of the male gametophyte and in the egg cell of the female gametophyte. Transgenic lines in which GEX3 was down-regulated or overexpressed, using the Arabidopsis GEX2 promoter, had reduced seed set. Reciprocal crosses and imaging after pollination with a reporter line showed that, in both cases, the defect causing reduced seed set occurred in the female. In the antisense lines, micropylar pollen tube guidance failed. In the overexpression lines, fertilization of mutant ovules was mostly blocked because pollen tube guidance failed, although, occasionally, non-viable embryos were formed. We conclude that properly regulated expression of GEX3 in the egg cell of Arabidopsis is essential for pollen tube guidance.

Double Fertilization in Arabidopsis thaliana Involves a Polyspermy Block on the Egg but Not the Central Cell

Scott, R. J., Armstrong, S. J., Doughty, J., Spielman, M. — 2008-05-21

In animal reproduction, thousands of sperm may compete to fertilize a single egg, but polyspermy blocks prevent multiple fertilization that would otherwise lead to death of the embryo. In flowering plants, successful seed development requires that only two sperm are delivered to the embryo sac, where each must fertilize a female gamete (egg or central cell) to produce the embryo and endosperm. Therefore, polyspermy must be avoided, not only to prevent abnormalities in offspring, but to ensure double fertilization. It is not understood how each sperm fertilizes only one female gamete, nor has the existence of polyspermy barriers been directly tested in vivo. Here, we sought evidence for polyspermy blocks in angiosperms using the polyspermic tetraspore (tes) mutant of Arabidopsis, which allows in-vivo challenge of egg and central cell with multiple male gametes. We show that tes mutant pollen tubes can transmit more than one sperm pair to an embryo sac, and that sperm from more than one pair can participate in fertilization. We detected endosperms but not embryos with ploidies that could only result from multiple fertilization. Our results therefore demonstrate an in-vivo polyspermy block on the egg, but not the central cell of a flowering plant.

A Dialogue between the Sirene Pathway in Synergids and the Fertilization Independent Seed Pathway in the Central Cell Controls Male Gamete Release during Double Fertilization in Arabidopsis

Rotman, N., Gourgues, M., Guitton, A.-E., Faure, J.-E., Berger, F. — 2008-05-16

Angiosperms sexual reproduction involves interactions between the two female gametes in the embryo sac and the two male gametes released by the pollen tube. The two synergids of the embryo sac express the FERONIA/SIRÈNE receptor-like kinase, which controls the discharge of the two sperm cells from the pollen tube. FER/SRN may respond to a ligand from the pollen tube. Alternatively, the interaction between FER/SRN and a ligand from the embryo sac may lead to a state of competence of the synergids allowing pollen tube discharge. Here, we report the new mutant scylla (syl) impaired in the control of pollen tube discharge. This mutant also produces autonomous endosperm development in absence of fertilization—a trait associated with the FERTILIZATION INDEPENDENT SEED (FIS) mutant class. This led us to identify autonomous endosperm in srn mutants and to demonstrate synergistic interactions between srn and the fis mutants. In addition, the fis mutants display defects in pollen tube discharge as in srn and syl mutants, confirming the interaction between the two pathways. Our findings suggest that pollen tube discharge is controlled by an interaction between the synergids expressing SRN/FER and the central cell expressing FIS genes.