Harwood J. Cranston

Characterization of cDNA clones for differentially expressed genes in embryos of dormant and nondormant Avena fatua L. caryopses.

The molecular regulation of seed dormancy was investigated using differential display to visualize and isolate cDNAs representing differentially expressed genes during early imbibition of dormant and nondormant Avena fatua L. embryos. Of about 3000 cDNA bands examined, 5 cDNAs hybridized with mRNAs exhibiting dormancy-associated expression patterns during the first 48 h of imbibition, while many more nondormancy-associated cDNAs were observed. Dormancy-associated clone AFD1 hybridized with a 1.5 kb mRNA barely detectable in dry dormant and nondormant embryos that became more abundant in dormant embryos after 24 h of imbibition. Clone AFD2 hybridized with two mRNAs, a 1.3 kb message constitutively expressed in dormant and nondormant embryos and a 0.9 kb message present at higher levels in dormant embryos after 3 h of imbibition. Nondormancy-associated clones AFN1, AFN2 and AFN3 hybridized with 1.5 kb, 1.7 kb and 1.1 kb mRNAs, respectively, that were more abundant in nondormant embryos during imbibition. Expression patterns of some mRNAs in dormant embryos induced to germinate by GA3 treatment were different than water controls, but were not identical to those observed in nondormant embryos. DNA sequence analysis revealed 76% sequence identity between clone AFN3 and a Citrus sinensis glutathione peroxidase-like cDNA, while significant sequence similarities with known genes were not found for other clones. Southern hybridization analyses showed that all clones represent low (1 to 4) copy number genes.

Dicamba resistance in kochia

Abstract Kochia plants resistant (R) to field rates of dicamba were characterized for their frequency of occurrence and levels of resistance and for the physiological fate of applied 14C-dicamba. Of 167 randomly sampled fields and seven fields identified by producers to contain R kochia, 19 contained plants that produced 1% or more R progeny. The maximum percentage of R progeny produced by parental plants from any field was 13%. An inbred R line derived from a field collection was 4.6-fold more resistant to dicamba than an inbred susceptible (S) line. Rates of 14C-dicamba uptake and translocation were similar in R and susceptible (S) plants up to 168 h after treatment (HAT). Concentrations of the primary metabolite, 5-hydroxy dicamba, were similar in R and S tissues up to 60 HAT, although amounts were significantly greater in R treated leaves by 96 and 168 HAT. However, because there were negligible levels of dicamba metabolites in R shoots and because the rate of metabolism was relatively slow, the observed changes were inadequate to account for observed resistance levels. Thus, dicamba resistance in kochia cannot be attributed to differential herbicide absorption, translocation, or metabolism. These findings, together with our field observations on the unusually slow spread of resistance within and among fields may indicate that dicamba resistance is a quantitative trait. Nomenclature: Dicamba; kochia, Kochia scoparia (L.) Schrad KCHSC.

Factors influencing Agrobacterium tumefaciens-mediated transformation and regeneration of the safflower cultivar ‘centennial’

The effects of co-cultivation conditions on transformation efficiency and direct shoot regeneration from seedling explants of safflower cv. ‘Centennial’ were examined. Agrobacterium tumefaciens strain EHA105/p35SGUSInt was more infective than LBA4404/pBI121 as determined by numbers of sectors expressing β-glucuronidase activity. Compared to nontransformed controls, efficiency of direct shoot regeneration was markedly decreased by co-cultivation with EHA105 and the decrease exacerbated by addition of acetosyringone, indicating that a hypersensitive response to bacterial infection may reduce organogenetic potential. Likewise exposure of co-cultivated explants to kanamycin or geneticin in selective media reduced regeneration efficiency. Addition of 500 mg l-1 carbenicillin slightly increased numbers of regenerating shoots. Tranfformed shoots were obtained only when kanamycin selection was initiated 1 or 2 days after co-cultivation. Presence of transgenes in geneticin-resistant shoots was confirmed using polymerase chain reaction and Southern hybridization assays.

Ciliary neurotrophic factor is expressed in the magnocellular neurosecretory system of the rat in vivo: Evidence for injury- and activity-induced upregulation

Although ciliary neurotrophic factor (CNTF) has been shown to promote the survival of magnocellular neurons when applied exogenously to explants of the paraventricular and supraoptic nuclei (SON) in vitro, little is known regarding its expression or regulation in the adult magnocellular neurosecretory system (MNS) following injury in vivo. Therefore, we utilized in situ hybridization and immunocytochemical analysis in conjunction with quantitative optical densitometric analysis to identify the cellular source of CNTF and examine the temporal pattern of its expression, following unilateral transection of the hypothalamo-neurohypophysial tract in the adult rat. In intact rats, CNTF immunoreactivity (CNTF-ir) was predominantly localized within identified astrocytes within the ventral glial limitans subjacent to the SON. Quantitative optical densitometric analysis of CNTF-ir levels in the axotomized SON demonstrated that the proportional area of CNTF-ir was significantly elevated between 3 and 30 days following injury. A significant but more limited increase was also observed in the non-injured contralateral SON. In situ hybridization confirmed the expression and upregulation of CNTF in the axotomized SON. These results demonstrate the expression of CNTF in the adult rodent MNS in vivo and provide evidence that levels of CNTF are upregulated in response to both direct injury, and heightened metabolic activity, within the lesioned and sprouting SON, respectively.

Dicamba-responsive genes in herbicide-resistant and susceptible biotypes of kochia (Kochia scoparia)

Abstract The herbicide resistance to dicamba (HRd) biotype of kochia is resistant to several auxinic herbicides and is impaired in shoot gravitropism and other auxin-mediated responses. To better characterize the biotype and investigate its mechanism of resistance, we used messenger RNA (mRNA) differential display to compare patterns of dicamba-induced gene expression in HRd and susceptible (S1) plants. More than 60,000 complementary DNA fragments were generated and examined, 106 of which were isolated and used as probes on Northern blots to confirm gene expression levels. Steady-state levels of > 90% of mRNAs did not change after dicamba application. However, several mRNAs were detected whose levels were decreased, increased, or differentially regulated between the biotypes within minutes of dicamba treatment. The abundance of three mRNAs decreased after treatment, two of which had significant sequence similarity to choline monooxygenase and 5,10-methylenetetrahydrofolate reductase, respectively. Conversely, increased expression levels were observed for a putative chloride channel protein, 1-aminocyclopropane-1-carboxylate synthase, and an unknown gene. Genes differentially expressed between HRd and S1 plants included those similar to a putative translation initiation factor, xyloglucan endotransglycosylase, and a hypothetical protein cloned from several organisms. The results demonstrate that mRNA differential display is a useful technique for discovering genes that are rapidly regulated as part of a physiological response, and that this approach may provide insight into the mechanism of auxinic herbicide resistance in kochia. Nomenclature: Dicamba; kochia, Kochia scoparia L. Schrad. KCHSC.

Wound-induced ethylene and germination of embryos excised from dormant Avena fatua L. caryopses.

Intact caryopses of dormant AN265 and M73 wild oat (Avena fatua L.) lines did not germinate when imbibed in water at 14°C, but germinated after exposure to ethylene during imbibition. Embryos excised from dormant caryopses and imbibed in water germinated readily. However, inhibitors of ethylene synthesis ([aminooxy]acetic acid and 2-[aminoethoxyvinyl] glycine) and ethylene action (2,5-norbornadiene and silver thiosulfate) delayed or almost completely inhibited germination of excised embryos. Embryos removed from inhibitor treatments after 15 d and incubated in water germinated normally. Except for 2,5-norbornadiene, inhibitors did not reduce germination of intact nondormant (afterripened) caryopses or embryos excised from nondormant caryopses. Reduced germination rates of embryos excised from dormant caryopses and incubated in 2,5-norbornadiene and 2-(aminoethoxyvinyl) glycine were reversed by applications of 0.05 μL/L ethylene. The results indicate that wound-induced ethylene synthesis may be responsible for germination of embryos excised from dormant wild oat caryopses.

Isolation and characterization of a cDNA encoding a sar-like monomeric GTP-binding protein in Avena fatua L.☆

Abstract Differential display of mRNAs from embryos of Avena fatua L. caryopses was used to isolate an mRNA more abundant in nondormant than dormant caryopses during early imbibition. The DNA sequence of the corresponding 579 bp cDNA, termed Af SAR1, is 92% identical to an Arabidopsis cDNA encoding the monomeric GTP-binding protein sar1p. Predicted amino acid sequences of the four conserved GTP binding and hydrolysis domains in Af SAR1 are 100% identical to sar1p. Af SAR1 mRNA levels increased 6-fold or more than 10-fold in nondormant embryos during the first 48 h of imbibition in water or GA3, respectively. However, mRNA levels increased only slightly and transiently in dormant embryos imbibed in water. mRNA abundance was highest in meristematic and actively growing tissues of A. fatua seedlings. Af SAR1 belongs to a small (two to four members) gene family as judged by Southern hybridizations. Increased abundance of this mRNA during early germination and in actively growing tissues indicates that the respective protein is associated with rapid cell elongation, cell division and growth.

CNTF receptor alpha is expressed by magnocellular neurons and expression is upregulated in the rat supraoptic nucleus during axonal sprouting

Ciliary neurotrophic factor (CNTF) is expressed by glial cells at multiple levels of the magnocellular neurosecretory system (MNS). CNTF is present in astrocytes in the hypothalamic supraoptic nucleus (SON) as well as in perivascular cells in the neurohypophysis, and a several fold increase in CNTF immunoreactivity occurs in the SON following either axotomy of magnocellular neurons or during axonal sprouting by intact magnocellular neurons. CNTF also promotes survival and stimulates process outgrowth from magnocellular neurons in vitro. While these findings suggest that CNTF may act as a growth factor in support of neuronal plasticity in the MNS, little is known regarding possible expression of receptors for CNTF in the MNS. We have therefore used immunocytochemistry and in situ hybridization to examine the expression of CNTF receptor alpha (CNTFRalpha) in the rat MNS. Robust immunoreactivity for CNTFRalpha was observed associated with oxytocinergic and vasopressinergic neurons distributed throughout the SON. Astrocytes located within the ventral glial lamina (VGL) of the SON were also immunoreactive for CNTFRalpha. Robust hybridization of an anti-sense [(35)S]-cRNA probe to CNTFRalpha mRNA was observed throughout the SON, while binding of a control sense probe was much lower. Grains were found clustered predominantly over neuronal somata, indicative of expression by magnocellular neurons within the SON. We next examined changes in expression of CNTFRalpha mRNA by magnocellular neurons 7 days following unilateral transection of the hypothalamo-neurohypophysial tract. The level of CNTFRalpha mRNA was increased 32% (compared to age-matched intact controls; p<0.05) in magnocellular neurons in the SON contralateral to the lesion, which are undergoing extensive collateral axonal sprouting, but was unchanged in axotomized magnocellular neurons in the SON ipsilateral to the lesion. These findings suggest that CNTF produced by MNS glia and acting via CNTFRalpha may exert neurotrophic effects on magnocellular neurons.