Regulating transgenic crops sensibly: lessons from plant breeding, biotechnology and genomics.

The costs of meeting regulatory requirements and market restrictions guided by regulatory criteria are substantial impediments to the commercialization of transgenic crops. Although a cautious approach may have been prudent initially, we argue that some regulatory requirements can now be modified to reduce costs and uncertainty without compromising safety. Long-accepted plant breeding methods for incorporating new diversity into crop varieties, experience from two decades of research on and commercialization of transgenic crops, and expanding knowledge of plant genome structure and dynamics all indicate that if a gene or trait is safe, the genetic engineering process itself presents little potential for unexpected consequences that would not be identified or eliminated in the variety development process before commercialization. We propose that as in conventional breeding, regulatory emphasis should be on phenotypic rather than genomic characteristics once a gene or trait has been shown to be safe.

Genomics applications to biotech traits: a revolution in progress?

Twenty years since the inception of the agricultural biotechnology era, only two products have had a significant impact in the market place: herbicide-resistant and insect-resistant crops. Additional products have been pursued but little success has been achieved, principally because of limited understanding of key genetic intervention points. Genomics tools have fueled a new strategy for identifying candidate genes. Primarily thanks to the application of functional genomics in Arabidopsis and other plants, the industry is now overwhelmed with candidate genes for transgenic intervention points. This success necessitates the application of genomics to the rapid validation of gene function and mode of action. As one example, the development of C-box binding factors (CBFs) for enhanced freezing and drought tolerance has been rapidly advanced because of the improved understanding generated by genomics technologies.

Regulation of disease resistance pathways by AP2/ERF transcription factors.

The AP2 transcription factor family, found only in plants, includes several genes that encode proteins involved in the regulation of disease resistance pathways. These genes are members of the ethylene response factor (ERF) subfamily of AP2 transcription factor genes, which have only a single DNA-binding domain and are distinct from members of the dehydration-responsive element binding (DREB) subfamily. Some ERF subgroups are enriched in such genes, suggesting that they have conserved functions that are required for the regulation of disease resistance pathways. The expression of several ERF genes is regulated by plant hormones, such as jasmonic acid, salicylic acid and ethylene, as well as by pathogen challenge. A phylogenetic overview of these genes, with a focus on Arabidopsis, rice and tomato, suggests that despite broad conservation of their function in monocots and dicots, some structural elements are specialized within each of these two lineages.

Arabinose substitution degree in xylan positively affects lignocellulose enzymatic digestibility after various NaOH/H2SO4 pretreatments in Miscanthus.

Xylans are the major hemicelluloses in grasses, but their effects on biomass saccharification remain unclear. In this study, we examined the 79 representative Miscanthus accessions that displayed a diverse cell wall composition and varied biomass digestibility. Correlation analysis showed that hemicelluloses level has a strong positive effect on lignocellulose enzymatic digestion after NaOH or H(2)SO(4) pretreatment. Characterization of the monosaccharide compositions in the KOH-extractable and non-KOH-extractable hemicelluloses indicated that arabinose substitution degree of xylan is the key factor that positively affects biomass saccharification. The xylose/arabinose ratio after individual enzyme digestion revealed that the arabinose in xylan is partially associated with cellulose in the amorphous regions, which negatively affects cellulose crystallinity for high biomass digestibility. The results provide insights into the mechanism of lignocellulose enzymatic digestion upon pretreatment, and also suggest a goal for the genetic modification of hemicelluloses towards the bioenergy crop breeding of Miscanthus and grasses.

Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-limited acres.

Commercially improved crop performance under drought conditions has been challenging because of the complexity of the trait and the multitude of factors that influence yield. Here we report the results of a functional genomics approach that identified a transcription factor from the nuclear factor Y (NF-Y) family, AtNF-YB1, which acts through a previously undescribed mechanism to confer improved performance in Arabidopsis under drought conditions. An orthologous maize transcription factor, ZmNF-YB2, is shown to have an equivalent activity. Under water-limited conditions, transgenic maize plants with increased ZmNF-YB2 expression show tolerance to drought based on the responses of a number of stress-related parameters, including chlorophyll content, stomatal conductance, leaf temperature, reduced wilting, and maintenance of photosynthesis. These stress adaptations contribute to a grain yield advantage to maize under water-limited environments. The application of this technology has the potential to significantly impact maize production systems that experience drought.

Inverted repeat of a heterologous 3'-untranslated region for high-efficiency, high-throughput gene silencing.

This report describes a method for the easy generation of inverted repeat constructs for the silencing of genes of unknown sequence which is applicable to high-throughput studies. This improved procedure for high-efficiency gene silencing is specific for a target gene, but does not require inverted repeat DNA of the target gene in the construct. The method employs an inverted repeat of the 3'-untranslated region (3'-UTR) of a heterologous gene, and has been demonstrated using the 3'-UTR region of the nopaline synthase (nos) gene from Agrobacterium tumefaciens, which is often used as the 3'-UTR for transgene constructs. In a population of independent tomato primary transformants harboring a stably integrated polygalacturonase (PG) transgene driven by a constitutive promoter and linked to an inverted repeat of the nos 3'-UTR, 51 of 56 primary transformants (91% of the population) showed highly effective post-transcriptional silencing of the PG gene, with PG mRNA abundance in ripe fruit reduced by 98% or more. The method was also effective in Arabidopsis, where two different, relatively uncharacterized plant transcription factors were also targeted effectively. This method has the advantage of ease and rapidity in preparation of the constructs, since a gene of interest can be inserted into a binary vector already containing the promoter and the inverted nos domain in a single-cloning step, and does not require any knowledge of the DNA sequence. The approach is suitable for high-throughput gene silencing studies, where it is necessary to investigate the function of hundreds to thousands of uncharacterized genes.

A novel, two-component system for cell lethality and its use in engineering nuclear male-sterility in plants.

Ablation of cells by the controlled expression of a lethal gene can be used to engineer plant traits such as male sterility and disease resistance. However, it may not be possible to achieve sufficient specificity of expression to prevent secondary effects in non-targeted tissues. In this paper we demonstrate that the extracellular ribonuclease, barnase, can be engineered into two complementary fragments, allowing overlapping promoter specificity to be used to enhance targeting specificity. Using a transient system, we first show that barnase can be split into two inactive peptide fragments, that when co-expressed can complement each other to reconstitute barnase activity. When a luciferase reporter gene was introduced into plant cells along with genes encoding both partial barnase peptides, a substantial reduction in luciferase activity was seen. Cytotoxicity of the reconstituted barnase was demonstrated by crossing together parents constitutively expressing each of the barnase fragments, then assaying their progeny for the presence of both partial barnase genes. None of over 300 tomato seeds planted resulted in a viable progeny that inherited both transgenes. When expression of the partial barnase genes was instead targeted to the tapetum, male sterility resulted. All 13 tomato progeny that inherited both transgenes were male sterile, whereas the three progeny inheriting only the N-terminal barnase gene were male fertile. Finally, we describe how male sterility generated by this type of two-component system can be used in hybrid seed production.