Hmg-coA reductase gene family in cotton (Gossypium hirsutum L.): unique structural features and differential expression of hmg2 potentially associated with synthesis of specific isoprenoids in developing embryos.

As a first step towards understanding the biosynthesis of isoprenoids that accumulate in specialized pigment glands of cotton at the molecular level, two full-length genes (hmg1 and hmg2) were characterized encoding hmg-coA reductase (HMGR; EC 1.1.1.34), the enzyme that catalyzes the formation of a key isoprenoid precursor. Cotton hmgr genes exhibited features typical of other plant genes, however, hmg2 encodes the largest of all plant HMGR enzymes described to date. HMG2 contains several novel features that may represent functional specialization of this particular HMGR isoform. Such features include a unique 42 amino acid sequence located in the region separating the N-terminal domain and C-terminal catalytic domain, as well as an N-terminal hydrophobic domain that is not found in HMG1 or other HMGR enzymes. DNA blot analysis revealed that hmg1 and hmg2 belong to small subfamilies that probably include homeologous loci in allotetraploid cotton (Gossypium hirsutum L.). Ribonuclease protection assays revealed that hmg1 and hmg2 are differentially expressed in a developmentally- and spatially-modulated manner during morphogenesis of specialized terpenoid-containing pigment glands in embryos. Induced expression of hmg2 coincided with a possible commitment to sesquiterpenoid biosynthesis in developing embryos, although other developmental processes also requiring HMGR cannot be excluded.

Factors affecting transient gene expression in protoplasts isolated from very slowly growing embryogenic callus cultures of wheat (Triticum aestivum L.).

Protoplasts isolated from embryogenic ('Mustang' and 'Chinese Spring') and non-embryogenic ('Mit') calli of wheat (Triticum aestivum L.) genotypes transiently expressed ß-glucuronidase (GUS) activity when electroporated with a plasmid containing the GUS gene and driven by an enhanced 35S promoter and a TMV leader sequence. Conditions for the maximum expression of GUS activity were: electroporation of the freshly isolated protoplasts at 250 Vcm(-1) and 250 µF for 2 s using 50 µg/ml of plasmid DNA; incubation of the protoplasts with the plasmid before the pulse for 2 h; and a 15-min recovery period on ice after the pulse. In general, a higher GUS activity was obtained in protoplasts of non-embryogenic (NE) callus origin than in those of embryogenic (E) callus origin. Only GUS constructs containing a duplicate 35S promoter derivative resulted in a significant level of GUS expression. The presence of the TMV viral leader sequence in the pAGUS1-TN2 plasmid construct resulted in a significant increase of GUS activity in the electroporated protoplasts of both callus types. On the other hand, protoplasts electroporated with the Adh1 promoter and intron showed a threefold less GUS activity than those electroporated with pAGUS1-TN2. Optimized conditions for DNA uptake and expression were very similar for protoplasts of both callus types. The importance of these findings for the successful regeneration of transgenic and fertile wheat plants is discussed.

Effects of cycling temperatures on fiber metabolism in cultured cotton ovules.

The effects of temperature on rates of cellulose synthesis, respiration, and long-term glucose uptake were investigated using cultured cotton ovules (Gossypium hirsutum L. cv Acala SJ1). Ovules were cultured either at constant 34 degrees C or under cycling temperatures (12 h at 34 degrees C/12 h at 15-40 degrees C). Rates of respiration and cellulose synthesis at various temperatures were determined on day 21 during the stage of secondary wall synthesis by feeding cultured ovules with [(14)C]glucose. Respiration increased between 18 and approximately 34 degrees C, then remained constant up to 40 degrees C. In contrast, the rate of cellulose synthesis increased above 18 degrees C, reached a plateau between about 28 and 37 degrees C, and then decreased at 40 degrees C. Therefore, the optimum temperature for rapid and metabolically efficient cellulose synthesis in Acala SJ1 is near 28 degrees C. In ovules cycled to 15 degrees C, respiration recovered to the control rate immediately upon rewarming to 34 degrees C, but the rate of cellulose synthesis did not fully recover for several hours. These data indicate that cellulose synthesis and respiration respond differently to cool temperatures. The long-term uptake of glucose, which is the carbon source in the culture medium, increased as the low temperature in the cycle increased between 15 and 28 degrees C. However, glucose uptake did not increase in cultures grown constantly at 34 degrees C compared to those cycled at 34/28 degrees C. These observations are consistent with previous observations on the responses of fiber elongation and weight gain to cycling temperatures in vitro and in the field.

Cultured Ovules as Models for Cotton Fiber Development under Low Temperatures.

Cotton fibers (Gossypium hirsutum L.) developing in vitro responded to cyclic temperature change similarly to those of field-grown plants under diumal temperature fluctuations. Absolute temperatures and rates of temperature change were similar under both conditions. In vitro fibers exhibited a "growth ring" for each time the temperature cycled to 22 or 15 degrees C. Rings were rarely detected when the low point was 28 degrees C. The rings seemed to correspond to alternating regions of high and low cellulose accumulation. Fibers developed in vitro under 34 degrees C/22 degrees C cycling developed similarly to constant 34 degrees C controls, but 34 degrees C/22 degrees C and 34 degrees C/15 degrees C cycling caused delayed onset and prolonged periods of elongation and secondary wall thickening. Control fiber length and weight were finally achieved under 34 degrees C/22 degrees C cycling, but both parameters were reduced at the end of the experiment under 34 degrees C/15 degrees C cycling. Fibers developed under all conditions had equal bundle tensile strength. These results demonstrate that: (a) cool temperature effects on fiber development are at least partly fiber/ovule-specific events; they do not depend on whole-plant physiology; and (b) cultured ovules are valid models for research on the regulation of the field cool temperature response.

In vitro selection and regeneration of cotton resistant to high temperature stress.

Cell suspension cultures of cotton (Gossypium hitirsutum L. cv. Coker 312) were exposed to various temperature:time treatments in order to select cell lines resistant to high temperature stress. Cells were exposed to 45°C for 3 h each day until the total accumulated hours of stress were: 0 h, 10 h, 75 h, 100 h, or 105 h (81 h pulsed then 24 h continuous). After the stress treatments, the cells were plated onto embryo development medium and plants were recovered. The embryogenic calli that were recovered were subcultured monthly for 6 months and tested for increased resistance to the temperature:time treatments previously determined to be lethal and to water stress as imposed by PEG. All of the selected cell lines were more resistant to both types of stress than the control cell lines. Leaf tissue of stress selected (Ro) formed and maintained callus growth when incubated at 38°C; whereas, tissue excised from nonselected controls rarely formed callus and calli which did form quickly became necrotic. These cells and plants will provide a tool for determining the mechanisms involved in resistance to high temperature stress.

Genotype specificity of the somatic embryogenesis response in cotton.

Thirty eight cultivars, strains, and races ofGossypium were screened for somatic embryogenesis with the protocols developed as a model forG. hirsutum L. cv. Coker 312. Four classes of response were identified; high, moderate, low, and non-embryogenic. Four cultivars were further screened with 13 growth regulator regimes to determine if culture environment could change the classification or induce a higher level of response. The classification or level of response did not change. Screening of individual seedlings within a cultivar indicated that genotypic variation for embryogenesis existed. Highly embryogenic individuals were selected from cvs. Coker 312 and Paymaster 303 for use as germplasm sources for transfer of the embryogenic trait to other cultivars and genetic stocks. Only genetically responsive genotypes are amenable to the model developed for Coker 312.

Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.).

Tissue culture methods for improvement of cotton has lagged seriously compared to other major crops. A method for regeneration of cotton which includes a morphogenetically competent cell suspension was needed to facilitate selection of stress-resistant variants and gene manipulation. Preliminary screening of eight strains of Gossypium hirsutum L. for embryogenic potential resulted in the production of somatic embryos in all strains. Coker 312 was selected for use in the development of a model regeneration system for G. hirsutum. Calli were initiated from hypocotyl tissues of 3-day-old-seedlings. Globular embryos were present after six weeks in culture. Calli were subcultured to liquid suspension in growth regulator-free medium. After three to four weeks, suspensions were sieved to collect globular and heart stage embryos. Collected embryos developed further when plated onto semi-solid medium. To induce germination and plantlet growth, mature embryos were placed on sterile vermiculite saturated with medium. Upon development of roots and two true leaves, plantlets were potted in peat and sand, and hardened. Mature plants and progeny have been obtained with this procedure. A high percentage of infertile plants was observed among the regenerants.