David B. Walden

Sequence, identification and characterization of cDNAs encoding two different members of the 18 kDa heat shock family of Zea mays L.

Heat-shocked maize seedlings (cv. Oh43) synthesize a characteristic set of heat-shock proteins (hsps) which include an 18 kDa family containing at least six major isoelectric variants. A cDNA library was constructed from poly(A)+ RNAs isolated from the radicles of heat-shocked maize seedlings and screened with a DNA fragment from the theoretical open reading frame of a putative Black Mexican Sweet maize hsp 18 genomic clone. Two clones, cMHSP18-3 and cMHSP18-9, were isolated, and the RNA transcripts generated from them were translated into proteins which immunoreact with antibodies directed against the maize 18 kDa hsps and exhibit the same electrophoretic characteristics as two different members of the 18 kDa hsp family. Nucleotide sequence analyses of the cDNAs in these clones reveal that their 5′ and 3′ untranslated regions exhibit 33–34% identity and that their protein encoding regions share 93% identity. The deduced amino acid sequences of these clones show 90% identity, and the apparent molecular masses and isoelectric points of these proteins agree with those established for two different 18 kDa hsps, numbered 3 and 6. This report substantiates that at least two of the 18 kDa hsps in maize are products of different but related genes. Moreover, it establishes that transcripts for these proteins accumulate during heat shock and that both their nucleotide and deduced amino acid sequences share extensive similarities with the class VI small hsps in soybean and with transcripts expressed during meiosis in Lilium.

Anther development of maize (Zea mays) and longstamen rice (Oryza longistaminata) revealed by cryo-SEM, with foci on locular dehydration and pollen arrangement.

AbstractKey messagePollen maturation in Poaceae. Another development has been extensively examined by various imaging tools, including transmission electron microscopy, scanning electron microscopy, and light microscopy, but none is capable of identifying liquid water. Cryo-scanning electron microscopy with high-pressure rapid freeze fixation is excellent in preserving structures at cellular level and differentiating gas- versus liquid-filled space, but rarely used in anther study. We applied this technique to examine anther development of Poaceae because of its economic importance and unusual peripheral arrangement of pollen. Maize and longstamen rice were focused on. Here, we report for the first time that anthers of Poaceae lose the locular free liquid during late-microspore to early pollen stages; the majority of pollen grains arranged in a tight peripheral whorl develops normally and reaches maturity in the gas-filled loculus. Occasionally, pollen grains are found situated in the locular cavity, but they remain immature or become shrunk at anthesis. At pollen stage, microchannels and cytoplasmic strands are densely distributed in the entire pollen exine and intine, respectively, suggesting that nutrients are transported into the pollen from the entire surface. We propose that in Poaceae, the specialized peripheral arrangement of pollen grains is crucial for pollen maturation in the gas-filled loculus, which enables pollen achieving large surface contact area with the tapetum and neighboring grains to maintain sufficient nutrient flow. This report also shows that the single aperture of pollen in Poaceae usually faces the tapetum, but other orientation is also common; pollen grains with different aperture orientations show no morphological differences.

Long-term cryogenic storage of Neurospora crassa spores☆

Abstract Neurospora crassa on agar slants in ampules were frozen by direct immersion into liquid nitrogen and stored at −196 °C for 0–5.5 years. The survival, estimated as percentage germination of conidia, after warming, varied between 81.1 and 97.6%. Although there was a slight decline in survival immediately after the freezing treatment, no further significant decline has been detected during 5.5 years storage. The regression coefficients, (0.43, control, and 0.97, liquid nitrogen storage for 5.5 years), do not predict extinction of viability.

Maize seedlings show cell-specific responses to heat shock as revealed by expression of RNA and protein.

The cellular localization of heat-shock proteins has been described in a number of experimental animal systems but is not well defined in plant systems. Sense and antisense RNA transcripts from the open reading frame (ORF) of 18-kDa maize heat-shock protein genes were employed in in situ hybridizations of inbred Oh43 radicles and plumules to reveal the locations of their mRNAs. Localization of the specific mRNAs to the younger meristematic cells of the root-tips and shoot-tips and also to the densely cytoplasmic cells of the vasculature was observed routinely. The ORF of one of our 18-kDa genes was cloned into an expression vector, and the 161-amino acid polypeptide was used to raise antibodies. Using a Fast Red procedure, the cellular positions of the heat-shock protein-antibody conjugates were observed in sections similar to those employed in the antisense RNA in situ hybridizations. The localization of the antibody appears to parallel closely the patterns of distribution of the mRNAs.

3-Dimensional Visualization of na2/na2 Stem in Maize

The phenotype of nana2 mutant (na2/na2, Fig 6) in maize (Zea mays L.) is characterized by its stubby dwarf appearance. As reported earlier, the longitudinal sections of stem of na2/na2 generally appear similar to the wild-type but with significant by shorter internodes [1] However, in the 2001 summer nursery (an excellent growing season) in London, Ontario, Canada, we observed an interesting characteristic of the mutant in more than 60 homozygous na2/na2 plants. This phenotypic expression was also observed in two plants previously in our 1999 and 1998 summer crop (relatively poor growing sessions).

The Study of Airflow Pattern Around a Maize Plant by Schlieren Optics

Pollen dispersion in maize is highly depend on the airflow patter around the plant. At dehiscence, anther swings about the elongated filament and the pollens are shed through the now basally oriented pore. Pollens are shed from anthers due to the vibration caused by the air current. It is our interest to understand the air flow pattern around a maize plant, in particular the tassel. Schlieren optics is an ideal system to visualize the air flow pattern. The system reveals distortion of the wavefronts due to density gradients within the volume of interest [1]. In order to study the airflow pattern around a maize plant, a small wind tunnel was constructed (Fig. 3). Three 16-inch box fans were stacked in series to provide different airflow velocities. Laminar flow was achieved by using a honeycomb panel constructed by using 5mm McDonald plastic beverage straws (Fig. 4). A Kestrel 3000 anemometer (Nielsen-Kellerman, Chester, PA) was used to determine the air velocity. Two 15cm spherical mirrors (f=150cm 1/10λ) were used to construct a Schlieren optical set-up (Figs. 1 and 3). A 100W Hg short arc lamp was used as the light source; a Sony camcorder operated in night-vision mode with built-in IR illuminator disabled was used to acquire the Schlieren images [2]. In addition, a second system using two f/3.5 Zeiss achormate lenses was constructed (Fig. 2), the system provides a beam diameter of 7cm for higher resolution views. Acetone or Freon vapor was ejected from a set of specially made nozzles in the air stream (Fig. 4). The acetone/Freon vapor has a different reflective index from surrounding air, hence it can be easily delineated from the surrounding air. Plants were placed downstream from the nozzle in the wind tunnel, and rotated to allow a study of multiple wind directions.