Tracey L. Reynolds

Impact of Genetics and Environment on the Metabolite Composition of Maize Grain

This study sought to assess genetic and environmental impacts on the metabolite composition of maize grain. Gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) measured 119 identified metabolites including free amino acids, free fatty acids, sugars, organic acids, and other small molecules in a range of hybrids derived from 48 inbred lines crossed against two different tester lines (from the C103 and Iodent heterotic groups) and grown at three locations in Iowa. It was reasoned that expanded metabolite coverage would contribute to a comprehensive evaluation of the grain metabolome, its degree of variability, and, in principle, its relationship to other compositional and agronomic features. The metabolic profiling results established that the small molecule metabolite pool is highly dependent on genotypic variation and that levels of certain metabolite classes may have an inverse genotypic relationship to each other. Different metabolic phenotypes were clearly associated with the two distinct tester populations. Overall, grain from the C103 lines contained higher levels of free fatty acids and organic acids, whereas grain from the Iodent lines were associated with higher levels of amino acids and carbohydrates. In addition, the fold-range of genotype mean values [composed of six samples each (two tester crosses per inbred x three field sites)] for identified metabolites ranged from approximately 1.5- to 93-fold. Interestingly, some grain metabolites showed a non-normal distribution over the entire corn population, which could, at least in part, be attributed to large differences in metabolite values within specific inbred crosses relative to other inbred sets. This study suggests a potential role for metabolic profiling in assisting the process of selecting elite germplasm in biotechnology development, or marker-assisted breeding.

Characterization of Unique Small RNA Populations from Rice Grain

Small RNAs (∼20 to 24 nucleotides) function as naturally occurring molecules critical in developmental pathways in plants and animals [1], [2]. Here we analyze small RNA populations from mature rice grain and seedlings by pyrosequencing. Using a clustering algorithm to locate regions producing small RNAs, we classified hotspots of small RNA generation within the genome. Hotspots here are defined as 1 kb regions within which small RNAs are significantly overproduced relative to the rest of the genome. Hotspots were identified to facilitate characterization of different categories of small RNA regulatory elements. Included in the hotspots, we found known members of 23 miRNA families representing 92 genes, one trans acting siRNA (ta-siRNA) gene, novel siRNA-generating coding genes and phased siRNA generating genes. Interestingly, over 20% of the small RNA population in grain came from a single foldback structure, which generated eight phased 21-nt siRNAs. This is reminiscent of a newly arising miRNA derived from duplication of progenitor genes [3], [4]. Our results provide data identifying distinct populations of small RNAs, including phased small RNAs, in mature grain to facilitate characterization of small regulatory RNA expression in monocot species.

Composition of Forage and Grain from Second-Generation Insect-Protected Corn MON 89034 Is Equivalent to That of Conventional Corn (Zea mays L.)

Insect-protected corn hybrids containing Cry insecticidal proteins derived from Bacillus thuringiensis have protection from target pests and provide effective management of insect resistance. MON 89034 hybrids have been developed that produce both the Cry1A.105 and Cry2Ab2 proteins, which provide two independent modes of insecticidal action against the European corn borer ( Ostrinia nubilalis ) and other lepidopteran insect pests of corn. The composition of MON 89034 corn was compared to conventional corn by measuring proximates, fiber, and minerals in forage and by measuring proximates, fiber, amino acids, fatty acids, vitamins, minerals, antinutrients, and secondary metabolites in grain collected from 10 replicated field sites across the United States and Argentina during the 2004-2005 growing seasons. Analyses established that the forage and grain from MON 89034 are compositionally comparable to the control corn hybrid and conventional corn reference hybrids. These findings support the conclusion that MON 89034 is compositionally equivalent to conventional corn hybrids.

Absorption, localisation, translocation and activity of glyphosate in barnyardgrass (Echinochloa crus-galli (L) Beauv) : influence of herbicide and surfactant concentration

The influence of sub-lethal and lethal doses of glyphosate (5 µg and 10 µg per plant) applied to the fourth leaf of barnyardgrass (Echinochloa crus-galli) was examined over a treatment period of up to 14 days. Assessments were undertaken on plant growth, chlorophyll fluorescence, absorption and translocation of [14C]glyphosate. Electronic autoradiography and image analysis were used to examine the distribution of [14C]glyphosate over the duration of the study. Major sinks affecting glyphosate distribution included the emerging fifth leaf, the roots and ‘shoot’ (meristem area). At 10 µg per plant, chlorophyll fluorescence declined over the treatment period; in the source and sink leaves effects were particularly evident at 5 DAT. Absorption and translocation of [14C]glyphosate (5 and 10 µg per plant) was rapid during 1–2 DAT, remaining relatively constant thereafter. Approximately 70% of the application was absorbed and, of this, 70% was translocated. The concentration of glyphosate increased in the sinks (the emerging fifth leaf, the roots and shoot (meristem) area) to a maximum at 3 DAT, thereafter declining. This decline was coincident with a decrease (2–3 DAT) in the level of photosynthesis (fluorescence) in the source and sink leaves of plants treated with 10 µg glyphosate. Incorporation of the surfactant MON 0818 at 0.5, 1.0 or 2.0 ml litre−1 enhanced herbicidal activity, absorption, translocation and sink accumulation of [14C]glyphosate (5 µg per plant), with absorption and translocation greatest at 0.5 ml litre−1 at 5 DAT. Herbicidal activity at 12–14 DAT, however, was greatest at the 1.0 ml litre−1 concentration. © 2000 Society of Chemical Industry

Mechanisms of glyphosate toxicity in velvetleaf (Abutilon theophrasti medikus)

In velvetleaf source leaves, glyphosate caused gradual inhibition of photosynthesis that increased over several days and was nearly complete by five days. Gas exchange measurements revealed that a decrease in stomatal conductance was a major factor in this reduction. Calvin cycle metabolite levels diminished along with photosynthesis rates but to a lesser extent than stomatal conductance, as indicated by the rise in water use efficiency and the lowering of internal leaf carbon dioxide. Though accumulation of shikimate confirmed that glyphosate was being taken up by the source leaves, this was insufficient to explain the lessened effect on photosynthesis. The protracted inhibition of photosynthesis allowed for greater translocation of glyphosate to sink tissues where it inflicted substantial damage. Consequently, sink tissue processes were more susceptible to disruption by glyphosate than were source leaf processes. The data also support the view that death of velvetleaf tissues was a result of restriction of water availability to the shoot induced by lethal disruption of root processes. The mechanism of prolonged plant death generated by sink tissue toxicity in velvetleaf contrasts with the mechanism observed in sugar beet, where death occurs rapidly due to the inhibition of source leaf processes.

Endogenous small RNAs in grain: Semi-quantification and sequence homology to human and animal genes

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effector molecules of RNA interference (RNAi), a highly conserved RNA-based gene suppression mechanism in plants, mammals and other eukaryotes. Endogenous RNAi-based gene suppression has been harnessed naturally and through conventional breeding to achieve desired plant phenotypes. The present study demonstrates that endogenous small RNAs, such as siRNAs and miRNAs, are abundant in soybean seeds, corn kernels, and rice grain, plant tissues that are traditionally used for food and feed. Numerous endogenous plant small RNAs were found to have perfect complementarity to human genes as well as those of other mammals. The abundance of endogenous small RNA molecules in grain from safely consumed food and feed crops such as soybean, corn, and rice and the homology of a number of these dietary small RNAs to human and animal genomes and transcriptomes establishes a history of safe consumption for dietary small RNAs.