Distribution and chemical forms of copper in the root cells of castor seedlings and their tolerance to copper phytotoxicity in hydroponic culture.

The subcellular localization and chemical forms of copper in castor (Ricinus communis L.) seedlings grown in hydroponic nutrient solution were identified by chemical extraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. The wild castor seeds were harvested from an abandoned copper mine in Tonglu Mountain, Daye City of Hubei Province, China. The results revealed that (1) the seedlings grew naturally in MS liquid medium with 40.00 mg kg(-1) CuSO4, in which the seedling growth rate and biomass index were 0.14 and 1.23, respectively, which were the highest values among all the treatments. The copper content in castor seedlings increased along with elevated CuSO4 concentration in the medium, reaching a maximum value of 16 570.12 mg kg(-1)(DW) when exposed to 60.00 mg L(-1) CuSO4, where 91.31% of the copper was accumulated in roots. (2) The copper existed in various chemical forms in the roots of the castor seedlings. Copper of 67.66% was extracted from the components of cell walls, such as exchangeable acidic polar compounds, cellulose and lignin, protein and pectin, and less concentrated in cell cytoplasm and nuclei. (3) Furthermore, the root cell walls were thickened when the castor seedlings exposed to CuSO4, with a large amount of high-density electron bodies, attached to the thickened cell walls. In the cell walls, most copper was bound to the carboxyl (-COOH) and hydroxyl (-OH) groups of acidic polar compounds, cellulose, hemicellulose, and polysaccharides. The conclusion showed that castor exhibited a strong tolerance to copper, the copper were accumulated mainly in the root cell, the root cell walls of castor were the major location of patience and detoxification in copper stress.

Putative calcium channels CchA and MidA play the important roles in conidiation, hyphal polarity and cell wall components in Aspergillus nidulans.

Although the high affinity Ca(2+) channel, Cch1, and its subunit Mid1 have been investigated and evaluated in yeast and some of filamentous fungi, little is known about the function of their homologs in the Aspergilli. Here, we have functionally characterized the yeast homologs, CchA and MidA, in Aspergillus nidulans using conditional and null deletion mutants. CchA and MidA not only have functional benefits of fast growth, which is consistent with Cch1 and Mid1 in yeast, but also have unique and complex roles in regulating conidiation, hyphal polarity and cell wall components in low-calcium environments. The defect of CchA or MidA resulted in a sharp reduction in the number of conidiospores, accompanied by abnormal metulae, and undeveloped-phialides at a higher density of inoculum. Most interestingly, these conidiation defects in mutants can, remarkably, be rescued either by extra-cellular Ca(2+) in a calcineurin-dependent way or by osmotic stress in a calcineurin-independent way. Moreover, the fact that the phenotypic defects are not exacerbated by the presence of the double deletion, together with the Y2H assay, indicates that CchA and MidA may form a complex to function together. Our findings suggest that the high-affinity Ca(2+) channel may represent a viable and completely unexplored avenue to reduce conidiation in the Aspergilli.

The important role of actinin-like protein (AcnA) in cytokinesis and apical dominance of hyphal cells in Aspergillus nidulans.

The actin cytoskeleton is involved in many processes in eukaryotic cells, including interaction with a wide variety of actin-binding proteins such as the actin-capping proteins, the actin filament nucleators and the actin cross-linking proteins. Here, we report the identification and characterization of an actinin-like protein (AcnA) from the filamentous fungus Aspergillus nidulans. Not only did the depletion of AcnA by alcA(p) promoter repression or the deletion of AcnA result in explicit abnormalities in septation and conidiation, but also the acnA mutants induced a loss of apical dominance in cells with dichotomous branching, in which a new branch was formed by splitting the existing tip in two. Consequently, the colony showed flabellate edges. Moreover, we found that the localization of the GFP-AcnA fusion was quite dynamic. In the isotropic expansion phase of the germinated spore, GFP-AcnA was organized as cortical patches with cables lining the cell wall. Subsequently, GFP-AcnA was localized to the actively growing hyphal tips and to the sites of septation in the form of combined double contractile rings. Our data suggest that AcnA plays an important role in cytokinesis and apical dominance of hyphal cells, possibly via actin-dependent polarization maintenance and medial ring establishment in A. nidulans. This is the first report, to our knowledge, of the function of an actinin-like protein in filamentous fungi.

Depletion of the MobB and CotA complex in Aspergillus nidulans causes defects in polarity maintenance that can be suppressed by the environment stress.

Polarized growth is a central feature in eukaryotes. Establishment and maintenance of cell polarity are coordinated by signaling pathways. In this study, we have identified MobB is required for the regulation of cell polarity in Aspergillus nidulans. Depletion of MobB by alcA (p) promoter repression or deletion of MobB abolished conidiation completely, and induced severe growth defects. mobB mutants showed abnormal nuclear segregation with increased number of nuclei in spores, but the formation of septa occurred among dividing cells. The phenotype of mobB in A. nidulans is similar to that of cotA. Furthermore, we verified that MobB interacted with CotA to function as a complex. Interestingly, both mobB and cotA deletion mutants clearly exhibited filament elongation by using environmental osmotic stress in the media. However, calcium channel blocker or chelator inhibited phenotype suppression of mobB or cotA mutants. These results suggest that Ca2+ is potentially involved in the response to the suppression coupled with osmotic stabilizer. This is the first report of the function of MobB in A. nidulans. We propose that the MobB/CotA complex, a component in the conserved RAM-signaling pathway, serves an important role in cell morphogenesis.