Extragenic suppressors of a dominant masculinizing her-1 mutation in C. elegans identify two new genes that affect sex determination in different ways

Summary: The her-1 regulatory switch gene in C. elegans sex determination is normally active in XO animals, resulting in male development, and inactive in XX animals, allowing hermaphrodite development. The her-1(n695gf) mutation results in the incomplete transformation of XX animals into phenotypic males. We describe four extragenic mutations that suppress the masculinized phenotype of her-1(n695gf) XX. They define two previously undescribed genes, sup-26 and sup-27. All four mutations exhibit semidominance of suppression and by themselves have no visible effects on sex determination in otherwise genotypically wild-type XX or XO animals. Analysis of interactions with mutations in the major sex-determining genes show that sup-26 and sup-27 influence sex determination in fundamentally different ways. sup-26 appears to act independently of her-1 to negatively modulate synthesis or function of tra-2 in both XX and XO animals. sup-27 may play a role in X-chromosome dosage compensation and influence sex determination indirectly.     

Extragenic suppressors of paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that alter the inner dynein arms

We have analyzed extragenic suppressors of paralyzed flagella mutations in Chlamydomonas reinhardtii in an effort to identify new dynein mutations. A temperature-sensitive allele of the PF16 locus was mutagenized and then screened for revertants that could swim at the restrictive temperature (Dutcher et al. 1984. J. Cell Biol. 98:229-236). In backcrosses of one of the revertant strains to wild-type, we recovered both the original pf16 mutation and a second, unlinked suppressor mutation with its own flagellar phenotype. This mutation has been identified by both recombination and complementation tests as a new allele of the previously uncharacterized PF9 locus on linkage group XII/XIII. SDS-PAGE analysis of isolated flagellar axonemes and dynein extracts has demonstrated that the pf9 strains are missing four polypeptides that form the I1 inner arm dynein subunit. The primary effect of the loss of the I1 subunit is a decrease in the forward swimming velocity due to a change in the flagellar waveform. Both the flagellar beat frequency and the axonemal ATPase activity are nearly wild-type. Examination of axonemes by thin section electron microscopy and image averaging methods reveals that a specific domain of the inner arm complex is missing in the pf9 mutant strains (see accompanying paper by Mastronarde et al.). When combined with other flagellar defects, the loss of the I1 subunit has synergistic effects on both flagellar assembly and flagellar motility. These synthetic phenotypes provide a screen for new suppressor mutations in other loci. Using this approach, we have identified the first interactive suppressors of a dynein arm mutation and an unusual bypass suppressor mutation.     

The function of actin-binding proteins in pollen tube growth

Pollen tube growth is a key step in sexual reproduction of higher plants. The pollen tube is a typical example of tip-growing cells and shows a polarized cytoplasm. To develop and maintain polarized growth, pollen tubes need a carefully regulated actin cytoskeleton. It is well known that actin-binding proteins are responsible for the direct control of dynamic actin filaments and serve as a link between signal transduction pathways and dynamic actin changes in determining cellular architecture. Several of these classes have been identified in pollen tubes and their detailed characterisation is progressing rapidly. Here, we aim to survey what is known about the major actin-binding proteins that affect actin assembly and dynamics, and their higher-order organisation in pollen tube growth.     

Effects of anoxia on pollen tube growth and tube wall formation of Impatiens glandulifera

Summary When pollen of Impatiens glandulifera was cultured in aerated liquid medium for 1 h, 70% of the pollen grains germinated; these attained an average tube length of 1 mm. Subsequently, these aerobic growth conditions were changed to anaerobic by substituting a nitrogen inlet for the air inlet. As a result, the pollen tubes stopped elongating and burst. The ultrastructural changes which occurred upon inducing anoxia were studied with samples taken at 0 s, 45 s, and 4 min after changing the gas. Anoxia caused rapid and considerable changes in the ultrastructure of the dictyosome vesicles involved in cell wall formation. There was an increase in the osmiophyly of the vesicle content, and the presence of fibrillar material became apparent. Simultaneously, the fusion behavior of the dictyosome vesicles changed. Instead of the normal fusion of the dictyosome vesicles with the plasma membrane, there was a premature fusion of the vesicles with each other inside the cytoplasm that resulted in the formation of aggregates. Furthermore, the cell wall precursors that were excreted were not incorporated in their usual configuration into the growing cell wall. Instead of a smooth inner cell wall surface, irregular thickenings were formed.     

A mutation that increases the activity of nonsense suppressors in Escherichia coli.

Summary We have isolated a new mutation, ups, that amplifies the suppressor activity of all the nonsense suppressors we have tested so far at low but not at high temperature. The properties of ups make it a very useful tool to improve the systems of temperature sensitive suppressors thus far described. ups maps between 25 to 27 min on the E. coli genetic map (Bachmann et al., 1976) and has no suppressor activity of its own. Its effects on translational fidelity are not influenced by mutations for ribosomal drug resistance. Thus, ups is different from ram which exhibits cooperative control of translation with other ribosomal proteins. The possible functions of ups in the cell are discussed.     

GL3 encodes a bHLH protein that regulates trichome development in arabidopsis through interaction with GL1 and TTG1

Arabidopsis trichome development and differentiation is a well-studied model for plant cell-fate determination and morphogenesis. Mutations in TRANSPARENT TESTA GLABRA1 (TTG1) result in several pleiotropic defects including an almost complete lack of trichomes. The complex phenotype caused by ttg1 mutations is suppressed by ectopic expression of the maize anthocyanin regulator R. Here it is demonstrated that the Arabidopsis trichome development locus GLABRA3 (GL3) encodes an R homolog. GL3 and GLABRA1 (GL1) interact when overexpressed together in plants. Yeast two-hybrid assays indicate that GL3 participates in physical interactions with GL1, TTG1, and itself, but that GL1 and TTG1 do not interact. These data suggest a reiterated combinatorial model for the differential regulation of such diverse developmental pathways as trichome cell-fate determination, root hair spacing, and anthocyanin secondary metabolism.     

Metabolism and roles of phosphatidylinositol 3-phosphate in pollen development and pollen tube growth in Arabidopsis

Phosphoinositides play important roles in eukaryotic cells, although they constitute a minor fraction of total cellular lipids. Specific kinases and phosphatases function on the regulation of phosphoinositide levels. Phosphatidylinositol 3-phosphate (PtdIns3P), a molecule of phosphoinositides regulates multiple aspects of plant growth and development. In this article, we introduce and discuss the kinases and phosphatases involved in PtdIns3P metabolism and their roles in pollen development and pollen tube growth in Arabidopsis.     

Suppressors of an Arabidopsis thaliana phyB mutation identify genes that control light signaling and hypocotyl elongation.

Ambient light controls the development and physiology of plants. The Arabidopsis thaliana photoreceptor phytochrome B (PHYB) regulates developmental light responses at both seedling and adult stages. To identify genes that mediate control of development by light, we screened for suppressors of the long hypocotyl phenotype caused by a phyB mutation. Genetic analyses show that the shy (short hypocotyl) mutations we have isolated fall in several loci. Phenotypes of the mutants suggest that some of the genes identified have functions in control of light responses. Other loci specifically affect cell elongation or expansion.     

The growth of the pollen tube wall in Oenothera organensis.

The growth of the pollen tube wall of Oenothera is effected by the expulsion of fibrillar material from the cytoplasm into the developing wall. This material may also be seen in the cytoplasm, contained in membrane-bound vesicles. It is not clear how the content of the vesicles is discharged, but it appears not to involve the participation of microtubules. The source of the cytoplasmic fibrillar bodies depends upon the stage of development of the pollen tube. The earilest growth is derived from the inclusion into the wall of vesicles containing pre-formed materials present in the grain on pollination. During the next stage of growth the wall is derived from the content of double-membraned inclusions also present in the pollen. The content of the former vesicles is not so similar to the wall as the latter, but intermediates between the 2 types of vesicle may be seen in the cytoplasm, indicating that the former are formed from the latter. Most of the tube wall is derived from the products of dictyosomes in the pollen grain or tube. These dicytosomes are few in number and they must be exceedingly active. This, and the observation that dictyosome vesicles are frequently associated with banked complexes of mitochondria, indicates that some steps in the metabolism of the vesicular content, perhaps phosphorylation, take place distant from the dicytosomes. These different sources of fibrillar material presumably permit the rapid starting of tube growth, without any attendant metabolism. However, it would be impossible to include enough pre-formed wall material in the grain to enable the full growth of the tube, so once started, it seems that the tube then relies on the elaboration of simple reserves for the contruction of its wall. These reserves are likely to be held in the pollen, and may be the large numbers of starch grains characteristic of the pollen cytoplasm.     

Extragenic suppressors of the nimX2(cdc2) mutation of Aspergillus nidulans affect nuclear division, septation and conidiation

The Aspergillus nidulans NIMX(CDC2) protein kinase has been shown to be required for both the G(2)/M and G(1)/S transitions, and recent evidence has implicated a role for NIMX(CDC2) in septation and conidiation. While much is understood of its G(2)/M function, little is known about the functions of NIMX(CDC2) during G(1)/S, septation, and conidiophore development. In an attempt to better understand how NIMX(CDC2) is involved in these processes, we have isolated four extragenic suppressors of the A. nidulans nimX2(cdc2) temperature-sensitive mutation. Mutation of these suppressor genes, designated snxA-snxD for suppressor of nimX, affects nuclear division, septation, and conidiation. The cold-sensitive snxA1 mutation leads to arrest of nuclear division during G(1) or early S. snxB1 causes hyperseptation in the hyphae and sensitivity to hydroxyurea, while snxC1 causes septation in the conidiophore stalk and aberrant conidiophore structure. snxD1 leads to slight septation defects and hydroxyurea sensitivity. The additional phenotypes that result from the suppressor mutations provide genetic evidence that NIMX(CDC2) affects septation and conidiation in addition to nuclear division, and cloning and biochemical analysis of these will allow a better understanding of the role of NIMX(CDC2) in these processes.     

Extragenic suppressors of loss-of-function mutations in the aspergillus FlbA regulator of G-protein signaling domain protein.

We showed previously that two genes, fl bA and fadA, have a major role in determining the balance between growth, sporulation, and mycotoxin (sterigmatocystin; ST) production by the filamentous fungus Aspergillus nidulans. fadA encodes the alpha subunit for a heterotrimeric G-protein, and continuous activation of FadA blocks sporulation and ST production while stimulating growth. fl bA encodes an A. nidulans regulator of G-protein signaling (RGS) domain protein that antagonizes FadA-mediated signaling to allow development. To better understand FlbA function and other aspects of FadA-mediated growth control, we have isolated and characterized mutations in four previously undefined genes designated as sfaA, sfaC, sfaD, and sfaE (suppressors of flbA), and a new allele of fadA (fadAR205H), all of which suppress a fl bA loss-of-function mutation ( fl bA98). These suppressors overcome fl bA losses of function in both sporulation and ST biosynthesis. fadAR205H, sfaC67, sfaD82, and sfaE83 mutations are dominant to wild type whereas sfaA1 is semidominant. sfaA1 also differs from other suppressor mutations in that it cannot suppress a fl bA deletion mutation (and is therefore allele specific) whereas all the dominant suppressors can bypass complete loss of fl bA. Only sfaE83 suppressed dominant activating mutations in fadA, indicating that sfaE may have a unique role in fadA- fl bA interactions. Finally, none of these suppressor mutations bypassed fl uG loss-of-function mutations in development-specific activation.     

Molecular evidence that most RNAs required for germination and pollen tube growth are stored in the mature pollen grain in petunia

After landing on the stigma, the pollen grain germinates and elongates a tube to deliver its generative nuclei to the egg cell of the ovule. The molecular mechanisms involved in the drastic morphological changes in the pollen grain during this fertilization process remain largely unknown. In this study, the expression of 732 randomly selected genes in petunia pollen and pollen tubes was analyzed by microarray and quantitative PCR analyses. We found no evidence for up-regulation of any of these genes in the pollen tube. Our findings provide support at the gene level for the longstanding hypothesis that pollen germination and tube growth are not dependent on new RNA synthesis and that the large number of RNAs required for germination and tube growth are stored in mature pollen grains.     

持续激活型CIPK9在花粉管生长过程中的生物学功能及亚细胞定位分析

植物的花粉管生长是一个多因素参与的生理学过程,需要多种信号传导系统来引导植物细胞完成。钙离子作为第二信使,可以通过钙传感器CBLs激活下游的蛋白激酶CIPKs参与调控细胞的极性发育过程。该研究中 CIPK9 被确定为候选基因,其C端与绿色荧光蛋白(GFP)相融合,通过基因枪技术在烟草花粉中进行瞬时表达,观察对应的亚细胞定位及花粉管中诱导的表型。结果表明:(1)GFP标记的CIPK9定位于花粉管中高速运动的颗粒状细胞器,并可随胞质环流进行规律的运动,为进一步探究CIPK9的生物学功能,还构建了持续激活型CIPK9(CACIPK9)。(2)与全长CIPK9相比较,CACIPK9缺少C末端的调控区域,并在激酶区域的激活环中进行了点突变,从而表现出不受调控的持续高活性。(3)缺少C端调控区的CACIPK9表现出非特异性的亚细胞定位,即与GFP对照相同的胞内弥散定位,说明CIPK9的C末端调控区对于其在花粉管中的正确定位发挥重要的调控作用。另外,CACIPK9过表达可以引起花粉管的去极化生长表型。这表明CIPK9作为钙信号下游家族的一员参与了花粉管极性生长的相关过程,并对花粉管的生长具有一定的调控作用。     

The phospholipid flippase ALA3 regulates pollen tube growth and guidance in Arabidopsis

Pollen tube guidance regulates the growth direction and ovule targeting of pollen tubes in pistils, which is crucial for the completion of sexual reproduction in flowering plants. The Arabidopsis (Arabidopsis thaliana) pollen-specific receptor kinase (PRK) family members PRK3 and PRK6 are specifically tip-localized and essential for pollen tube growth and guidance. However, the mechanisms controlling the polar localization of PRKs at the pollen tube tip are unclear. The Arabidopsis P4-ATPase ALA3 helps establish the polar localization of apical phosphatidylserine (PS) in pollen tubes. Here, we discovered that loss of ALA3 function caused pollen tube defects in growth and ovule targeting and significantly affected the polar localization pattern of PRK3 and PRK6. Both PRK3 and PRK6 contain two polybasic clusters in the intracellular juxtamembrane domain, and they bound to PS in vitro. PRK3 and PRK6 with polybasic cluster mutations showed reduced or abolished binding to PS and altered polar localization patterns, and they failed to effectively complement the pollen tube-related phenotypes of prk mutants. These results suggest that ALA3 influences the precise localization of PRK3, PRK6, and other PRKs by regulating the distribution of PS, which plays a key role in regulating pollen tube growth and guidance.

AMINOPHOSPHOLIPID ATPASE3 guides pollen tubes by regulating the distribution of anionic phospholipids to affect the precise localization of certain pollen-specific receptor kinases at pollen tubes.

IN A NUTSHELL Background: In flowering plants, pollen tube guidance regulates the rapid growth and timely targeting of the pollen tube to the ovule in the pistil during sexual reproduction, when signaling between the male and female gametophytes occur. The small peptide-RLK signaling module is essential for the interaction between the male and female gametophyte. Certain members of the pollen-specific receptor kinase (PRK) family have different subcellular localization patterns in Arabidopsis pollen tubes and play critical roles in pollen tube growth and guidance. However, the molecular mechanisms that regulate and maintain the polar localization of PRKs at the pollen tube tip are still unknown. Question: We were interested in exploring how Arabidopsis P4-ATPase (aminophospholipid ATPase, ALA) precisely regulates pollen tube guidance and maintains the polar localization patterns of PRK6 and PRK3. How plant ALA family members regulate pollen tube guidance has not yet been documented. Findings: The loss of ALA3 function not only caused sluggish pollen tube growth and aberrant ovule targeting but also affected the polar localization patterns of several PRKs at the pollen tube tip. Members of the PRKs family can directly interact with anionic phospholipids such as phosphatidylserine (PS), and the capacity of PRK3/6 to bind anionic phospholipids is crucial for both their polar localization and physiological functions. ALA3 establishes and maintains the polar distribution of PS, which influences secretory vesicles-mediated polar trafficking at the pollen tube tip to affect the distribution of PRK3 and PRK6. On the other hand, PS might also directly recruit PRK3 and PRK6 to the pollen tube tip and sustain their localization. Next steps: The localization of PRKs is a complex, finely regulated process. The C-termini of PRKs may also affect their polar distribution. More research is required to reveal how the C-terminus domain precisely controls the localization of PRKs.     

Ion dynamics and hydrodynamics in the regulation of pollen tube growth

A coherent picture of pollen tube growth is beginning to emerge that couples ion dynamics with biochemical, biophysical and cytological processes in ordered and controlled feedback circuits that define the nature of polarized apical growth. It is a paradox, however, that complete understanding of the mechanical forces that drive cell elongation in this system still remains to be fully achieved. The results of our recent studies to characterize Cl^– ion dynamics during apical growth in tobacco pollen tubes led us to re-examine this question in the light of a possible force-generating role provided by hydrodynamic flow. Previously we found that oscillatory Cl^– efflux from the apex is closely coupled to oscillatory growth and the cell volume of the apical domain. Cl^– influx occurs in a region of the tube that is distal to the clear zone; hence, a vectorial flow of anion traverses the apical domain and fluxes out of the tip with oscillatory dynamics. Because of the effects that this could induce on charge and osmotic potentials, water could potentially flow through the apical domain, linked to the flux of Cl^–. This conjecture is consistent with studies in other plant cells that demonstrate a pivotal role for flux through anion channels in the control or normalization of osmotic status. In the current report, the relationship between Cl^– efflux oscillations and the physical characteristics of the apical dome during oscillatory growth is examined in closer detail. Evidence is presented that shows a cyclic deformation of the extreme apex occurs during the growth pulse and is correlated with cyclic Cl^– efflux. In addition, there is a dramatic increase in the number and density of clear thread-like zones traversing the apical plasma membrane during the process of tip elongation. Possible functional roles of Cl^– flux and hydrodynamics are discussed in the context of what drives tip elongation during cycles of pollen tube growth. Received: 23 November 2000 / Revision accepted: 19 June 2001