Daniel Z. Skinner

Alteration of antioxidant enzyme gene expression during cold acclimation of near isogenic wheat lines

Reactive oxygen species (ROS) are harmful to living organisms due to the potential oxidation of membranes, DNA, proteins, and carbohydrates. Freezing injury has been shown to involve the attack of ROS. Antioxidant enzymes can protect plant cells from oxidative stress imposed by freezing injury; therefore, cold acclimation may involve an increase in the expression of antioxidant enzymes. In this study, quantitative RT-PCR was used to measure the expression levels of antioxidant enzymes during cold acclimation in near-isogenic lines (NILs) of wheat, differing in the Vrn1-Fr1 chromosome region that conditions winter versus spring wheat growth habit. The antioxidant genes monitored were mitochondrial manganese superoxide dismutase (MnSOD), chloroplastic Cu,Zn superoxide dismutase (Cu,ZnSOD), iron superoxide dismutase (FeSOD), catalase (CAT), thylakoid-bound ascorbate peroxidase (t-APX), cytosolic glutathione reductase (GR), glutathione peroxidase (GPX), cytosolic mono-dehydroascorbate reductase (MDAR), chloroplastic dehydroascorbate reductase (DHAR). The expression levels were upregulated (MnSOD, MDAR, t-APX, DHAR, GPX, and GR), downregulated (CAT), or relatively constant (FeSOD and Cu,ZnSOD). The Vrn1-Fr1 region seemed to have a role in regulating the expression level of some of the antioxidant enzyme genes because t-APX, CAT and MnSOD expressed to significantly higher levels in the winter wheat NIL than the spring wheat NIL after 4 weeks’ cold acclimation.

Post-acclimation transcriptome adjustment is a major factor in freezing tolerance of winter wheat

Cold-acclimated winter wheat plants were slowly frozen to −10°C, and then the temperature was either maintained at −10°C or was lowered further to −12°C. Expression levels of a total of 423 genes were significantly altered in these treatments; genes upregulated outnumbered those downregulated by about a 9:1 ratio. Sixty-eight genes were upregulated at least fivefold in all freezing treatments; 17 of these 68 encoded transcription factors including C-repeat binding factor (Cbf), WRKY, or other Zn-finger proteins, indicating strong upregulation of genes involved in transcription regulation. Sixteen of the 68 highly upregulated genes encoded kinases, phosphatases, calcium trafficking-related proteins, or glycosyltransferases, indicating upregulation of genes involved in signal transduction. Six genes encoding chlorophyll a/b binding-like proteins were upregulated uniquely in response to the -12°C treatment, suggesting a protective role of pigment-binding proteins in freezing stress response. Most genes responded similarly in the very freezing tolerant cultivar Norstar and in the moderately freezing tolerant Tiber, but some genes responded in opposite fashion in the two cultivars. These results showed that wheat crowns actively adapt as the temperature declines to potentially damaging levels, and genetic variation for this ability exists among cultivars.

Differential expression of manganese superoxide dismutase sequence variants in near isogenic lines of wheat during cold acclimation

Numerous sequence variants of wheat (Triticum aestivum L.) manganese superoxide dismutase (MnSOD) genes have been found. Quantitative real-time PCR was used to measure the expression levels of three MnSOD genes distinguished by a variable amino acid, and three genes distinguished by sequence variation in the 3′ untranslated region (3′ UTR), in wheat plants grown at 20°C and cold-acclimated for 1–4 weeks at 2°C. The amino acid variants did not differ significantly in expression levels, however, differential expression of genes differing in the 3′ UTR was observed. Diploid wheat-related species also carried sequence variants of MnSOD, with differing levels of expression, suggesting diversification of the MnSOD gene family occurred prior to the polyploidization events of hexaploid wheat.

Expression of pigmentation genes following electroporation of albino Monascus purpureus

A UV-induced albino strain of Monascus purpureus was subjected to electroporation in the presence of genomic DNA from a wild-type red strain of the fungus. Eight colonies expressed color after several weeks of growth. The growth rates of all eight color variants were significantly greater than the recipient and donor strains under some culture conditions. Spectrophotometric analysis of the pigments extracted from the color variants revealed the pigments had absorbance spectra different from the DNA donor strain. These color variants may have resulted from transformation with wild-type DNA, mutation reversion, or activation of alternative pathway(s)—i.e., new mutations—that resulted in pigment production.

Genes Upregulated in Winter Wheat (Triticum aestivum L.) during Mild Freezing and Subsequent Thawing Suggest Sequential Activation of Multiple Response Mechanisms.

Exposing fully cold-acclimated wheat plants to a mild freeze-thaw cycle of −3°C for 24h followed by +3°C for 24 or 48h results in dramatically improved tolerance of subsequent exposure to sub-freezing temperatures. Gene enrichment analysis of crown tissue from plants collected before or after the −3°C freeze or after thawing at +3°C for 24 or 48h revealed that many biological processes and molecular functions were activated during the freeze-thaw cycle in an increasing cascade of responses such that over 150 processes or functions were significantly enhanced by the end of the 48 h, post-freeze thaw. Nearly 2,000 individual genes were upregulated more than 2-fold over the 72 h course of freezing and thawing, but more than 70% of these genes were upregulated during only one of the time periods examined, suggesting a series of genes and gene functions were involved in activation of the processes that led to enhanced freezing tolerance. This series of functions appeared to include extensive cell signaling, activation of stress response mechanisms and the phenylpropanoid biosynthetic pathway, extensive modification of secondary metabolites, and physical restructuring of cell membranes. By identifying plant lines that are especially able to activate these multiple mechanisms it may be possible to develop lines with enhanced winterhardiness.

Transformation of Monascus purpureus to hygromycin B resistance with cosmid pMOcosX reduces fertility

Albino strain KB20M1 of Monascus purpureus was genetically transformed to hygromycin B resistance with cosmid pMOcosX, using biolistic bombardment. Conidia and mycelial fragments were used as the recipient material and rupture disk strengths of 6.2 and 9.3 MPa each yielded a transformed isolate from five bombardments. Southern analysis suggested a single copy of the cosmid had integrated into each of the transformants. Both of the independent transformants formed cleistothecia but ascospore formation was greatly reduced or absent, suggesting the integration and expression of the genes carried on pMOcosX interfered with fertilization and/or ascospore formation in this homothallic fungus.

Quantitative real-time PCR method to detect changes in specific transcript and total RNA amounts

Quantitative real-time PCR (qRT-PCR), used in conjunction with reverse transcriptase, has been applied to the determination of the number of copies of a transcript per unit mass of RNA, but did not indicate any change in the amount of total RNA per mass of tissue. In the present work, we described a simple method to use qRT-PCR to estimate the change in the amount of total RNA per unit mass of wheat ( Triticum aestivum L.) tissue in response to cold temperature. Three qRT-PCR templates, i.e. control, cold-exposed, and one of RNA extracted from a sample consisting of equal masses of control and cold-exposed tissue, were analyzed. The number of copies of target transcript per unit mass of RNA was estimated from the three samples using standard qRT-PCR techniques. Equations describing the number of copies of the target sequence in each of the tissue samples were solved simultaneously to describe the relative proportion of the target sequence that originated from each tissue sample in the mixture, thereby providing an estimate of relative amounts of total RNA in the two tissues.

Molecular Cloning and Expression of Sequence Variants of Manganese Superoxide Dismutase Genes from Wheat

Reactive oxygen species (ROS) are very harmful to living organisms due to the potential oxidation of membrane lipids, DNA, proteins, and carbohydrates. transformed E.coli strain QC 871, superoxide dismutase (SOD) double-mutant, with three sequence variant MnSOD1, MnSOD2, and MnSOD3 manganese superoxide dismutase (MnSOD) gene isolated from wheat. Although all QC 871 transformants grown at 37 ℃ expressed mRNA of MnSOD variants, only MnSOD2 transformant had functional SOD activity. MnSOD3 expressed active protein when grown at 22℃, however, MnSOD1 did not express functional protein at any growing and induction conditions. The sequence comparison of the wheat MnSOD variants revealed that the only amino acid difference between the sequence MnSOD2 and sequences MnSOD1 and 3 is phenylalanine/serine at position 58 amino acid. We made MnSOD2S58F gene, which was made by altering the phenylalaine to serine at position 58 in MnSOD2. The expressed MnSOD2S58F protein had functional SOD activity, even at higher levels than the original MnSOD2 at all observed temperatures. These data suggest that amino acid variation can result in highly active forms of MnSOD and the MnSOD2S58F gene can be an ideal target used for transforming crops to increase tolerance to environmental stresses.