ALTERED ZYGOSPORE WALL ULTRASTRUCTURE CORRELATES WITH REDUCED ABIOTIC STRESS RESISTANCE IN A MUTANT STRAIN OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1

The zygospore of Chlamydomonas is a diploid resting stage that provides protection from environmental extremes. The remarkable abiotic stress resistance of the zygospore can be explained, in part, by the presence of a massive wall that includes a sporopollenin‐containing surface layer ( Van Winkle‐Swift and Rickoll 1997 ). A Chlamydomonas monoica Strehlow zygospore‐specific mutant strain (D19) was obtained previously by screening for loss of chloroform resistance in zygospore populations derived from self‐mating of post‐mutagenesis clones. Exposure of D19 zygospores to solar UV radiation or germicidal radiation also resulted in a pronounced decrease in survival of D19 zygospores relative to wildtype zygospore survival. Similarly, resistance to NaCl‐induced osmotic shock was reduced in D19 zygospores, especially when exposed to very high (e.g., 20% w/v) salt concentrations. Mature zygospores of C. monoica exhibit a UV‐induced blue surface autofluorescence that may indicate the presence of phenolic wall components. The intensity of zygospore autofluorescence was significantly reduced in D19 zygospores. As revealed by TEM, the surface layer of mature homozygous D19 zygospores was disrupted, suggesting a defect in wall assembly. Zygospore‐specific chloroform sensitivity, UV sensitivity, and reduced autofluorescence cosegregated in tetrads derived from D19 heterozygotes (i.e., if a progeny clone from a cross involving D19 and a normal strain was found to be chloroform sensitive, it was always also UV sensitive and showed reduced autofluorescence), indicating that all three characteristics were the consequence of the same Mendelian mutation.     

Variations of the Surface Sculpture and Cell Wall Ultrastructure of the Zygospores in Chlamydomonas geitleri (Chlorophyta)

The superficial cell wall ornamentation in the zygospores of the alga Chlamydomonas geitleri Ettl (Chlorophyta) is formed by thickenings of the cell wall which are shaped into a network of anastomosing ribs, sometimes with local wart-like protuberances. Clearly different sculpture patterns (given by presence, arrangement and/or morphological modification of sculpture elements) were accompanied by many transient forms. Sculpture variations occurred even in clonal cultures. In the zygospore cell wall of C. geitleri, the inner, outer and middle layer can be distinguished from the morphological point of view. The relatively thin outer (sporopollenin) layer covers the whole surface of the zygospore wall. The thicker inner layer adhering to the zygospore protoplast forms, either solely or together with the middle layer (possessing a fine meshwork substructure), variously shaped thickening of the zygospore cell wall. Discussed are the ultrastructural morphology of the cell wall in Chlamydomonas zygospores, the striking similarity of the cell wall ultrastructure of zygospores in C. geitleri to the ultrastructure of the cell wall of vegetative cells in some green algae (subfamily Scotiellocystoideae), as well as the extensive morphological variability of the zygospore wall sculpture in C geitleri and its species specificity.     

SELF-STERILE AND MATURATION-DEFECTIVE MUTANTS OF THE HOMOTHALLIC ALGA,CHLAMYDOMONAS MONOICA (CHLOROPHYCEAE).1

Homothallic sexual reproduction in Chlamydomonas monoica Strehlow culminated in the formation of mature, chloroform-resistant zygospores (zygotes) in clonal culture. Early in the zygote maturation process, a distinctive “primary zygote wall” was released into the culture medium where it remained stable for at least several days. This wall appeared as a rigid, darkly-outlined, and often multilayered structure, as viewed by phase contrast microscopy. From a sample, of 2500 individual clones isolated after ethyl methanesulfonate mutagenesis, five maturation-defective strains (zym) produced abnormal zygotes which failed to release a primary zygote wall, failed to develop the normal reticulate zygospore wall, and disintegrated within five days. These strains were utilized to identify additional mutants which were sexually competent, but self-sterile (het). Mixed cultures of the zym and het mutant strains were found to contain numerous, fully-matured, chloroform-resistant zygospores and discarded primary zygote walls. In combination, the two types of mutants provided a useful system for the selective recovery of heterozygous zygospores, thus facilitating genetic studies on a homothallic Chlamydomonas.     

First Discovery of Two Polyketide Synthase Genes for Mitorubrinic Acid and Mitorubrinol Yellow Pigment Biosynthesis and Implications in Virulence of Penicillium marneffei

Background

The genome of P. marneffei, the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia, possesses 23 polyketide synthase (PKS) genes and 2 polyketide synthase nonribosomal peptide synthase hybrid (PKS-NRPS) genes, which is of high diversity compared to other thermal dimorphic pathogenic fungi. We hypothesized that the yellow pigment in the mold form of P. marneffei could also be synthesized by one or more PKS genes.

Methodology/Principal Findings

All 23 PKS and 2 PKS-NRPS genes of P. marneffei were systematically knocked down. A loss of the yellow pigment was observed in the mold form of the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants. Sequence analysis showed that PKS11 and PKS12 are fungal non-reducing PKSs. Ultra high performance liquid chromatography-photodiode array detector/electrospray ionization-quadruple time of flight-mass spectrometry (MS) and MS/MS analysis of the culture filtrates of wild type P. marneffei and the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants showed that the yellow pigment is composed of mitorubrinic acid and mitorubrinol. The survival of mice challenged with the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants was significantly better than those challenged with wild type P. marneffei (P<0.05). There was also statistically significant decrease in survival of pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants compared to wild type P. marneffei in both J774 and THP1 macrophages (P<0.05).

Conclusions/Significance

The yellow pigment of the mold form of P. marneffei is composed of mitorubrinol and mitorubrinic acid. This represents the first discovery of PKS genes responsible for mitorubrinol and mitorubrinic acid biosynthesis. pks12 and pks11 are probably responsible for sequential use in the biosynthesis of mitorubrinol and mitorubrinic acid. Mitorubrinol and mitorubrinic acid are virulence factors of P. marneffei by improving its intracellular survival in macrophages.     

PKS1 and PKS2 affect the phyA state in etiolated Arabidopsis seedlings.

Autophosphorylation of phytochrome A (phyA) and transphosphorylation of its reaction partners, phytochrome kinase substrate 1 (PKS1) in particular, might play important functions in signal transduction from phyA. It was shown that PKS1 and PKS2 physically interact with phyA and phyB in vitro, and that overexpression of PKS1 interferes with phytochrome signaling in vivo. Moreover, both pks1 and pks2 loss of function mutants are specifically defective for one branch of phyA signaling. We therefore used in vivo fluorescence spectroscopy to test whether mutations in pks1 and pks2 or overexpression of PKS1 (PKS1OX) have an effect on phyA and its subpopulations, phyA' and phyA'. It was found that the emission spectra of phyA in all the Arabidopsis lines are similar. The phyA content in the single mutants pks1 and pks2, and also in PKS1OX, was 1.2-1.5 times higher than in the wild type, whereas the phyA'/phyA' ratio remained practically unchanged (approx. 1.0). However, in the double mutant pks1pks2, the picture is reversed--the phyA concentration remained unchanged, while the phyA'/phyA' ratio shifted dramatically towards phyA'(0.3). This suggests that (i) the changes in PKS1 or PKS2 content may affect the total phyA concentration, (ii) PKS1, together with PKS2, could be critical for the formation of phyA', thus shifting the equilibrium towards phyA' in the double mutant and (iii) these variations in the phyA' and phyA' content may contribute to the mutant phenotype of pks1, pks2 and PKS1OX. The fact that in the single mutants there are only small changes in the phyA'/phyA' ratio, while in the double mutant the ratio is considerably affected, indicates that PKS1 or PKS2 act redundantly with each other in this regard.     

Numerical and structural changes in dictyosomes during zygospore germination ofClosterium ehrenbergii

Summary Numerical and structural changes in dictyosomes during the germination of zygospores inClosterium ehrenbergii were examined by electron microscopy. In the dormant mature zygospores, two parallel cisternac were seen which were derived from the disorganization of dictyosomes during the maturation of zygospores. After the induction of germination, the two parallel cisternae developed into dictyosomes with ten or eleven cisternae. The dictyosomes doubled in number by division every day for four days and reached, at the time of germination, a density of distribution similar to that found in the youngest zygospore. On the 4th day after the induction of germination, dictyosomes produced two kinds of vesicles which appear to be involved in the formation of new cell wall layers. The germination of the zygospore was effected by the escape of the cell covered with the new cell wall layers through the broken old cell wall layers.     

SEXUAL PROCESSES AND PHYLOGENETIC RELATIONSHIPS OF A HOMOTHALLIC STRAIN IN THE CLOSTERIUM PERACEROSUM–STRIGOSUM–LITTORALE COMPLEX (ZYGNEMATALES,CHAROPHYCEAE)1

Members of the Closterium peracerosum–strigosum–littorale (C. psl.) complex are unicellular charophycean algae in which there are two modes of zygospore formation, heterothallic and homothallic. A homothallic strain of Closterium (designation, kodama20) was isolated from a Japanese rice paddy field. Based on alignment of the 1506 group‐I introns, which interrupt nuclear SSU rDNAs, homothallic kodama20 is most closely related to the heterothallic mating group II‐B, which is partially sexually isolated from group II‐A. Time‐lapse photography of the conjugation process in kodama20 revealed that most of the observed zygospores originated from one vegetative cell. The sexual conjugation process consisted of five stages: (1) cell division resulting in the formation of two sister gametangial cells from one vegetative cell, (2) formation of a sexual pair between the two sister gametangial cells (or between gametangial cells of another adjoined individual), (3) formation of conjugation papillae, (4) release of gametic protoplasts from both members of a pair, and (5) formation of the zygospore by protoplast fusion. For conjugation to progress, the cell density and light condition in the culture was critical. We suggested the presence of a conjugation promotion factor.     

Complementation and Preliminary Linkage Analysis of Zygote Maturation Mutants of the Homothallic Alga, CHLAMYDOMONAS MONOICA

Sexual reproduction in Chlamydomonas monoica is homothallic: pair formation and cell fusion occur in clonal culture and give rise to a heavily walled diploid zygospore. During maturation of the young zygote, a distinctive "primary zygote wall" is released before the development of the highly reticulate zygospore wall. Using ethyl methanesulfonate and ultraviolet irradiation as mutagens, we have isolated 19 maturation-defective (zym ) mutant strains which upon self-mating produce inviable zygotes. These zygotes fail to release a primary zygote wall, fail to develop the normal zygospore wall, and eventually undergo spontaneous lysis. In nearly all cases, the mutations appear to be expressed only in the diploid zygote; pleiotropic effects on vegetative cell growth or morphology are not evident.—Complementation testing performed on 17 of these mutants indicates that all are recessive and that they define seven distinct complementation groups. Preliminary tetrad analysis of two-factor and multifactor zym crosses provides no evidence for physical clustering of the maturation genes, and instead suggests that they are widely distributed throughout the nuclear genome.     

A fluorescence‐activated cell sorting‐based strategy for rapid isolation of high‐lipid Chlamydomonas mutants

There is significant interest in farming algae for the direct production of biofuels and valuable lipids. Chlamydomonas reinhardtii is the leading model system for studying lipid metabolism in green algae, but current methods for isolating mutants of this organism with a perturbed lipid content are slow and tedious. Here, we present the Chlamydomonas high‐lipid sorting (CHiLiS) strategy, which enables enrichment of high‐lipid mutants by fluorescence‐activated cell sorting (FACS) of pooled mutants stained with the lipid‐sensitive dye Nile Red. This method only takes 5 weeks from mutagenesis to mutant isolation. We developed a staining protocol that allows quantification of lipid content while preserving cell viability. We improved separation of high‐lipid mutants from the wild type by using each cell's chlorophyll fluorescence as an internal control. We initially demonstrated 20‐fold enrichment of the known high‐lipid mutant sta1 from a mixture of sta1 and wild‐type cells. We then applied CHiLiS to sort thousands of high‐lipid cells from a pool of about 60 000 mutants. Flow cytometry analysis of 24 individual mutants isolated by this approach revealed that about 50% showed a reproducible high‐lipid phenotype. We further characterized nine of the mutants with the highest lipid content by flame ionization detection and mass spectrometry lipidomics. All mutants analyzed had a higher triacylglycerol content and perturbed whole‐cell fatty acid composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than the wild type. The unprecedented throughput of CHiLiS opens the door to a systems‐level understanding of green algal lipid biology by enabling genome‐saturating isolation of mutants in key genes.     

拟南芥PKS5点突变体对盐碱胁迫的响应特性

拟南芥蛋白激酶PKS5参与植物对外界的盐碱胁迫信号响应过程.为探求PKS5不同结构域对外界盐碱胁迫下的响应功能,以PKS5点突变体pks5-2、pks5-4、pks5-5、pks5-6、pks5-7、pks5-8和pks5-9为材料,分析PKS5不同点突变体在外界盐碱胁迫下的特性.结果显示:(1)pks5-2、pks5-6、pks5-7和pks5-8对外界盐碱胁迫的主根生长表型与野生型存在差异,pks5-4、pks5-5和pk5-9则无差异,其中的pks5-2和pks5-8主根生长对盐碱胁迫表现出抗性表型,而pks5-6和pks5-7表现出敏感表型.(2)当PKS5发生点突变后其突变基因的表达发生改变,与野生型基因相比,PKS5-2、PKS5-6与PKS5-7的表达均有所降低.(3) PKS5点突变蛋白的亚细胞定位与野生型相比不存在差异,在细胞核、细胞质及细胞膜中均有分布.(4)pks5各点突变体内的Na+含量在野生型与点突变体间存在显著差异,pks5-2体内的Na+含量较野生型降低,而pks5-6和pks5-7体内的Na+则升高.研究表明,PKS5不同位置点突变导致植物对外界盐碱胁迫有着不同的响应过程,预示PKS5不同的结构域在其功能上存在差异.     

Jasmonate‐dependent modifications of the pectin matrix during potato development function as a defense mechanism targeted by Dickeya dadantii virulence factors

The plant cell wall constitutes an essential protection barrier against pathogen attack. In addition, cell‐wall disruption leads to accumulation of jasmonates (JAs), which are key signaling molecules for activation of plant inducible defense responses. However, whether JAs in return modulate the cell‐wall composition to reinforce this defensive barrier remains unknown. The enzyme 13–allene oxide synthase (13–AOS) catalyzes the first committed step towards biosynthesis of JAs. In potato (Solanum tuberosum), there are two putative St13–AOS genes, which we show here to be differentially induced upon wounding. We also determine that both genes complement an Arabidopsis aos null mutant, indicating that they encode functional 13–AOS enzymes. Indeed, transgenic potato plants lacking both St13–AOS genes (CoAOS1/2 lines) exhibited a significant reduction of JAs, a concomitant decrease in wound‐responsive gene activation, and an increased severity of soft rot disease symptoms caused by Dickeya dadantii. Intriguingly, a hypovirulent D. dadantii pel strain lacking the five major pectate lyases, which causes limited tissue maceration on wild‐type plants, regained infectivity in CoAOS1/2 plants. In line with this, we found differences in pectin methyl esterase activity and cell‐wall pectin composition between wild‐type and CoAOS1/2 plants. Importantly, wild‐type plants had pectins with a lower degree of methyl esterification, which are the substrates of the pectate lyases mutated in the pel strain. These results suggest that, during development of potato plants, JAs mediate modification of the pectin matrix to form a defensive barrier that is counteracted by pectinolytic virulence factors from D. dadantii.     

THE ZYGOSPORE WALL OF CHLAMYDOMONAS MONOICA (CHLOROPHYCEAE): MORPHOGENESIS AND EVIDENCE FOR THE PRESENCE OF SPOROPOLLENIN1

Chlamydomonas monoica Strehlow is being developed as a model for genetic analysis of zygospore morphogenesis, and many relevant mutant strains are available. To provide the basis for interpreting the ultrastructural phenotypes of zygospore mutants, an analysis of wall morphogenesis in wildtype zygospores of C. monoica was undertaken. Following synthesis of a thick, fibrous, primary zygote wall, granular material accumulated between the plasma membrane and the primary zygote wall and aggregated into a repetitive array of electron-opaque fibrous stripes. A new wall layer, the outer layer of the secondary zygospore wall, first appeared as segments with a fibrous outer surface overlying a well-defined band of electron-translucent material. These segments gave rise to an intact sheath adjacent to the plasma membrane. Beneath this sheath, electron-opaque material (forming the inner layer of the secondary zygospore wall) accumulated unevenly and forced the surface sheath to undulate, creating a pattern of peaks and valleys that was exposed to the external environment 4 rupture and release of the primary zygote wall. The zygospore wall included material resistant to degradation by potassium hydroxide, 2-aminoethanol, and acetolysis, but it was destroyed by exposure to chromic acid. These characteristics, in combination with the autofluorescence of untreated zygospore walls and their failure to stain with phloroglucinol, suggest that sporopollenin may be responsible for many of the resistant properties associated with the mature zygospore of Chlamydomonas.     

Self‐rescue of an EXTENSIN mutant reveals alternative gene expression programs and candidate proteins for new cell wall assembly in Arabidopsis

Plants encode a poorly understood superfamily of developmentally expressed cell wall hydroxyproline‐rich glycoproteins (HRGPs). One, EXTENSIN3 (EXT3) of the 168 putative HRGPs, is critical in the first steps of new wall assembly, demonstrated by broken and misplaced walls in its lethal homozygous mutant. Here we report the findings of phenotypic (not genotypic) revertants of the ext3 mutant and in‐depth analysis including microarray and qRT‐PCR (polymerase chain reaction). The aim was to identify EXT3 substitute(s), thus gaining a deeper understanding of new wall assembly. The data show differential expression in the ext3 mutant that included 61% (P ≤ 0.05) of the HRGP genes, and ability to self‐rescue by reprogramming expression. Independent revertants had reproducible expression networks, largely heritable over the four generations tested, with some genes displaying transgenerational drift towards wild‐type expression levels. Genes for nine candidate regulatory proteins as well as eight candidate HRGP building materials and/or facilitators of new wall assembly or maintenance, in the (near) absence of EXT3 expression, were identified. Seven of the HRGP fit the current model of EXT function. In conclusion, the data on phenotype comparisons and on differential expression of the genes‐of‐focus provide strong evidence that different combinations of HRGPs regulated by alternative gene expression networks, can make functioning cell walls, resulting in (apparently) normal plant growth and development. More broadly, this has implications for interpreting the cause of any mutant phenotype, assigning gene function, and genetically modifying plants for utilitarian purposes.     

The role of arabinokinase in arabinose toxicity in plants

Plant cell wall polymers are synthesized by glycosyltransferases using nucleotide sugars as substrates. Most UDP‐sugars are synthesized from UDP‐glucose via de novo pathways but salvage pathways work in parallel to recycle sugars, which have been released during cell wall polymer and glycoprotein turnover. Here we report on the cloning and biochemical analysis of two arabinokinases in Arabidopsis. Arabinokinase is a 100 kDa protein located in the cytosol with a putative N‐terminal glycosyltransferase domain and a C‐terminal sugar‐1‐kinase domain. This unique structure is highly conserved in the plant kingdom. Arabinokinase has a high affinity for l ‐arabinose, which is the only sugar substrate of this GHMP (galactose; homoserine; mevalonate; phosphomevalonate) kinase. Plants that were knocked‐out for arabinokinase and the previously described ara1‐1 mutant were characterized. The ARA1‐1 mutant form of the enzyme carries a point mutation in an α‐helix. The mutation is close to the substrate binding site and changes the K_m value for arabinose from 80 μm in the wild type to 17 000 μm in ARA1‐1. The previous arabinose toxicity explanation is challenged by knockout plants in arabinokinase that accumulate higher levels of arabinose but do not show signs of arabinose toxicity. Analysis of marker genes from sugar signalling pathways (SnRK1 and Tor) suggest that ara1‐1 misinterprets its carbon energy status. Although glucose is present in ara1‐1 similar to wild type levels, it constitutively changes gene expression as typically found in wild type plants only under starvation conditions. Furthermore, ara1‐1 shows increased expression of marker genes for programmed cell death as found in other lesion mimic mutants.     

Ultrastructural studies on zygomycotan fungi in the Zoopagaceae and Cochlonemataceae

The morphology of fungi in the Zoopagaceae and Cochlonemataceae (Zoopagales, Zoopagomycotina, Zygomycota) is reviewed, and some new ultrastructural information is added on conidia and zygospores, as well as haustoria in the former family and vegetative thalli in the latter. The cell wall of the conidia of Acaulopage dichotoma, Ac. tetraceros, Stylopage cephalote, Zoophagus insidians, and Zph. tentaclum (Zoopagaceae), and of Cochlonema odontosperma and Endocochlus gigas (Cochlonemataceae), is known to be composed of outer electron-dense and inner less dense layers in ultrathin sections, and no additional cell walls were found on the conidial cell wall. Although two nuclei were found in the zygosporangium before maturation to the zygospore in Acaulopage rhaphidospora (Zoopagaceae), more than one nucleus had never been observed previously in a zygospore in either of these families in ultrathin sections.     

The Polyketide Synthase Gene pks4 of Trichoderma reesei Provides Pigmentation and Stress Resistance

Species of the fungal genus Trichoderma (Hypocreales, Ascomycota) are well-known for their production of various secondary metabolites. Nonribosomal peptides and polyketides represent a major portion of these products. In a recent phylogenomic investigation of Trichoderma polyketide synthase (PKS)-encoding genes, the pks4 from T. reesei was shown to be an orthologue of pigment-forming PKSs involved in synthesis of aurofusarin and bikaverin in Fusarium spp. In this study, we show that deletion of this gene in T. reesei results in loss of green conidial pigmentation and in pigmentation alteration of teleomorph structures. It also has an impact on conidial cell wall stability and the antagonistic abilities of T. reesei against other fungi, including formation of inhibitory metabolites. In addition, deletion of pks4 significantly influences the expression of other PKS-encoding genes of T. reesei. To our knowledge, this is the first indication that a low-molecular-weight pigment-forming PKS is involved in defense, mechanical stability, and stress resistance in fungi.     

Crystal Structure of Mycobacterium tuberculosis Polyketide Synthase 11 (PKS11) Reveals Intermediates in the Synthesis of Methyl-branched Alkylpyrones

PKS11 is one of three type III polyketide synthases (PKSs) identified in Mycobacterium tuberculosis. Although many PKSs in M. tuberculosis have been implicated in producing complex cell wall glycolipids, the biological function of PKS11 is unknown. PKS11 has previously been proposed to synthesize alkylpyrones from fatty acid substrates. We solved the crystal structure of M. tuberculosis PKS11 and found the overall fold to be similar to other type III PKSs. PKS11 has a deep hydrophobic tunnel proximal to the active site Cys-138 to accommodate substrates. We observed electron density in this tunnel from a co-purified molecule that was identified by mass spectrometry to be palmitate. Co-crystallization with malonyl-CoA (MCoA) or methylmalonyl-CoA (MMCoA) led to partial turnover of the substrate, resulting in trapped intermediates. Reconstitution of the reaction in solution confirmed that both co-factors are required for optimal activity, and kinetic analysis shows that MMCoA is incorporated first, then MCoA, followed by lactonization to produce methyl-branched alkylpyrones.