Advancing rice research with cutting edge science |
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Symposium 236 27-29 Mar 2000 Held at the International Rice Research Institute, The Philippines Synopses A framework for sequencing the rice genome and first insights into genome organization Gernot Presting, Yeisoo Yu, Todd Wood, M Arief Budiman, Sung-Sick Woo, Ralph Dean, David Frisch, Rod Wing Rice is an important food crop and a model plant for other cereal genomes. The Clemson University Genomics Institute framework project, begun two years ago in anticipation of the now ongoing international effort to sequence the rice genome, is nearing completion. Two BAC libraries have been constructed from the Oryza sativa cultivar Nipponbare. Over 100 000 BAC end sequences have been generated from these libraries and, at a current total of 28 Mbp, represent 6.5% of the total rice genome sequence. This sequence information has allowed us to draw first conclusions about unique and redundant rice genomic sequences. In addition, more than 60 000 clones (19 genome equivalents) have been successfully fingerprinted and assembled into contigs using FPC software. Many of these contigs have been anchored to the rice chromosomes using a variety of techniques. Hybridization experiments have shown these contigs to be very robust. Contig assembly and hybridization experiments have revealed some surprising insights into the organization of the rice genome, which will have significant repercussions for the sequencing effort. Integration of BAC end sequence data with anchored contig information has provided unexpected revelations on sequence organization at the chromosomal level. Rice genomics: genetic and physical maps and the current status of genome sequencing Takashi Matsumoto, Jianzhong Wu, Tomoya Baba, Yuichi Katayose, Kimiko Yamamoto, Katsumi Sakata, Masahiro Yano and Takuji Sasaki Rice is one of the major crops in the world. Rice genomics is important not only for the food supply for the increasing world population but also for basic research in plant biology. The Rice Genome Research Program has set up the fundamentals for rice genome analysis: a cDNA catalogue, a genetic linkage map and a YAC-based physical map. In 1998, this project was reconstructed as a new program with three main goals: (1) complete genome sequencing, (2) elucidation of gene function, and (3) application of genome information to breeding. In this presentation, we introduce our optimized system for genome sequencing and describe the present status of genome sequencing of rice chromosomes 1 and 6. For rice genome sequencing, we have used the bacteriophage P1-derived artificial chromosome (PAC) genomic clones with 100 to 150 kb inserts as targets for shotgun assemblage. PAC clones are being located onto their chromosomal positions using physically positioned EST markers. The PAC DNA was then sheared into small fragments to create a shotgun subclone library and the subclone inserts were sequenced from both ends. Sequence data were assembled into PAC sequences, annotated and made public through a public database and our website. Rice as the pivotal cereal? The impact and applications of comparative genetics Mike Gale, Katrien Devos, Graham Moore It has become clear that gene content and gene order along plant chromosomes tend to be conserved over much greater periods than we previously imagined. Conservation of gene colinearity over the 60 million years of grass evolution is such that the genomes of all the major cereals can be described on a single consensus map. Application of these comparisons is aided by recent results on gene densities and evolutionary expansion of inter-genic chromosome regions. On the other hand the evidence for extensive duplication of genes and gene regions, even in model diploids such as rice and Arabidopsis, will complicate applications. The most immediate benefit to flow from these results is our ability to pool genetic, biochemical and physiological knowledge gained independently in the different cereals over the past century. Similarly �orphan� grass crops, e.g. foxtail millet, pearl millet and sugar cane, can rapidly gain status as �genetic organisms� by their incorporation into the consensus map. The presence, location and mode of action of key genes, such as the GA-insensitive dwarfing genes which have been so important in international wheat breeding, can be predicted across species. Key genes in large genome species, such as the genes controlling chromosome pairing in hexaploid bread wheat, should be accessible via the genomic DNA sequence in the colinear chromosome regions of the simpler rice genome. However, the more general success of these approaches will depend on the degree that colinearity is maintained at the sequence level. Finally we will review the extent to which colinearity can be expected to extend beyond the grasses, in particular to arabidopsis and the eudicots. These considerations will affect our future strategy for genomic sequencing and genomics research in crop plants. Development of a gene switch for rice Steve Goff A chemically regulated gene switch as well as a synthetic regulated target promoter has been developed and transformed into rice. Data will be presented that demonstrate the use of this transferred regulatory system in transgenic rice lines. A research gene switch and a field use gene switch system will be described, and experimental data presented on the use of these systems with synthetic target promoters driving reporter gene constructs. Such systems for chemically regulated gene expression are an integral part of a cereal functional genomics effort. These tools will also provide for control of plant gene expression in field situations. Introduction of genes encoding C4 photosynthesis into rice plants: physiological consequences Maurice S B Ku Using an Agrobacterium-mediated transformation system, we have introduced three C4 photosynthesis genes from maize into rice with high levels of expression. Transgenic rice plants overexpressing the maize phosphoenolpyruvate carboxylase (PEPC) exhibit a higher photosynthetic rate (up to 30%) and a reduced O2 inhibition of photosynthesis than untransformed plants. There is a small increase in the amount of atmospheric CO2 being directly fixed by PEPC. The increased photosynthetic capacity is mainly due to an enhanced stomatal conductance and a higher internal CO2 concentration. Similarly, transgenic rice plants overexpressing the maize pyruvate, orthophosphate dikinase (PPDK), also have higher photosynthetic rates (up to 35%) than untransformed plants, again due to an enhanced stomatal conductance. Using conventional hybridization, we have integrated the maize PEPC and PPDK genes into the same transgenic rice plants. In the segregation population, the photosynthetic rates of plants with high levels of both maize enzymes are twice higher than those of untransformed plants. Under full sunlight conditions, the photosynthetic capacity of field-grown PEPC transgenic rice plants is twice that of untransformed plants. Consistently, PEPC transgenic plants have a higher photosynthetic quantum yield by PSII and a higher capacity to dissipate excess energy photochemically and non-photochemically. Preliminary data from a field test show that the grain yield is 12% higher in PEPC and 35% higher in PPDK transgenic rice plants, relative to untransformed plants. Taken together, these results suggest that introduction of C4 photosynthesis enzymes into rice has a good potential to enhance its photosynthetic capacity and yield. Increasing rice photosynthesis by manipulation of the acclimation and adaptation to light Peter Horton Sunlight is the driving force for photosynthesis and biomass accumulation by plants, which is in turn an important determinant of crop yield. There are strong relationships between intercepted radiation and yield, and radiation conversion efficiency is viewed as a central parameter in considerations of crop improvement. On the other hand, in excess, sunlight can lead to impairment of photosynthetic performance, a process known as photoinhibition�most importantly when photosynthetic capacity is restricted by sub-optimal conditions the resulting abiotic stress is greatly amplified by light. There are two important considerations in assessing the interaction of plants with light: (a) under what conditions does photoinhibition occur?; (b) how does the plant respond to the light environment both in the short term (regulation) and in the long term (acclimation)? Despite a wealth of knowledge about these processes in model plant species, it is impossible to predict how significant they are in influencing the yield of rice. Therefore, in collaboration with IRRI, we have undertaken a study of photoinhibition and photoacclimation of rice under field conditions. The results of this study will be presented, along with an assessment of the implications for improvement of rice yield. Increasing rice productivity and yield by manipulation of starch synthesis Thomas W Okita, Jindong Sun, Chotipa Sakulringharoj, Sang-Bong Choi, Hiroyuki Ito and Gerald E Edwards Plant productivity and yield are dependent on source-sink relationships, i.e. the capacity of source leaves to fix CO2 and the capacity of developing sink tissues and organs to assimilate and convert this fixed carbon into dry matter. Studies from our laboratories as well as others have demonstrated that rice productivity and yield are mainly sink-limited during its development. Using non-evasive techniques, the effect of temperature, light, humidity, CO2 and O2 levels on CO2 fixation and photosystem II activity under steady-state and transient conditions was measured to determine whether the capacity to utilize the initial photosynthetic products (triose-P) is limiting and causing inhibition of photosynthesis. The results showed that photosynthesis in rice is limited by product utilization under moderate-to-high light, moderate temperature, and high humidity (i.e. under conditions commonly occurring in the field). This limitation in triose-P utilization, evident at both the vegetative and reproductive stages of rice development, may be associated with limited capacity for carbohydrate synthesis in rice leaves (which are poor accumulators of starch) or feedback due to limited sink strength of developing seeds. The strategies used to relieve these constraints on triose-P utilization by altering starch metabolism will be discussed. Dissection of resistance gene-mediated pathways in plants BJ Feys, MA Austin, C Rusterucci, LJ Moisan and JE Parker Disease resistance (R) genes are routinely introduced into high yielding crop varieties to improve resistance to agronomically damaging pathogens. Whilst the R gene-encoded products are recognitionally specific, interacting directly or indirectly with a corresponding pathogen avirulence (Avr) determinant, they are subject to diversifying selection pressures that may enhance the creation of new pathogen recognition capabilities. Genetic and molecular analyses in different plant species have also revealed resistance signalling components that provide further targets for manipulation in crop improvement strategies. A thorough understanding of the processes that regulate both plant�pathogen recognition and the induction of an appropriate resistance response should provide a fresh perspective on combating plant disease. Many recent studies have focused on the model plant, Arabidopsis, which is host to all the major pathogen classes. Mutational screens in Arabidopsis have identified genes that are required for R gene function, and analyses suggest that distinct signalling pathways are conditioned by R protein structure rather than pathogen type. Two such signalling components, EDS1 and PAD4, are required for the accumulation of salicylic acid, a phenolic molecule that functions in systemic immunity. The cloning, molecular and biochemical characterization of these components suggests processes that may be central to their resistance signalling function. Regulation of systemic acquired resistance by NPR1 and its partners Xinnian Dong, Xin Li, Yuelin Zhang, Weihua Fan, Mark Kinkema and Joseph Clarke The NPR1 protein of Arabidopsis thaliana has been shown to be an important regulatory component of systemic acquired resistance (SAR). Mutations in the NPR1 gene block the induction of SAR by the signal molecule salicylic acid (SA). NPR1 contains ankyrin repeats and a BTB domain which are involved in interaction with other protein(s). To study further the function of NPR1 and the regulatory mechanism of SAR, we have used both molecular and genetic approaches to identify additional SAR regulatory components. Through a yeast two-hybrid screen, we found that NPR1 interacts specifically with bZIP transcription factors. The involvement of bZIP transcription factors in controlling the SA-induced genes has been suggested by a number of promoter studies performed on these genes. It was found that the as1 element, which is a binding site for bZIP transcription factors, is essential for SA-induced gene expression. In a genetic screen for suppressors of npr1, we found a mutant, sni1, that restored the responsiveness to SAR induction in npr1. The genetic characteristics of the sni1 mutant and the sequence of SN11 protein is a negative regulator of SAR. We believe that SAR is controlled by both positive and negative regulators. Improving plant drought, salt and freezing tolerance by gene transfer of a single stress-inducible transcription factor Kazuko Yamaguchi-Shinozaki and Kazuo Shinozaki* Plant productivity is greatly affected by environmental stresses such as drought, salt loading and freezing. We reported that a cis-acting promoter element, the dehydration response element (DRE), plays an important role in regulating gene expression in response to these stresses in Arabidopsis. The transcripotion factor DREB1A specifically interacts with the DRE and induces expression of stress tolerance genes. We show here that overexpression of the cDNA encoding DREB1A in transgenic Arabidopsis plants activated the expression of many of theses stress tolerance genes under normal growing conditions and resulted in improved tolerance to drought, salt loading, and freezing. However, use of the strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive expression of DREB1A also resulted in severe growth retardation under normal growing conditions. In contrast, expression of DREB1A from the stress inducible rd29A promoter gave rise to minimal effects on plant growth while providing an even greater tolerance to stress conditions than did expression of the gene from the CaMV promoter. As the DRE-related regulatory element is not limited to Arabidopsis, the DREB1A cDNA and the rd29A promoter may be useful to improve the stress tolerance of agriculturally important crops by gene transfer. Strategies for dissection of defence response pathways in rice Jan E Leach, Hei Leung* and Guo-Liang Wang� The cloning of major resistance genes has led to a better understanding of the molecular biology of the steps for induction of resistance, yet much remains to be discovered about the downstream genes that collectively confer resistance, i.e. the defence response (DR) genes. We are dissecting the pathways contributing to resistance in rice by identifying a collection of mutants with deletions or other structural rearrangements in DR genes. Through database mining, a collection of 'candidate genes', i.e. putative DR genes, including a compilation of sequence information to generate gene-specific and gene-family-specific primers and probes, has been established. Additional DR genes are being selected by subtractive selection of cDNAs synthesized from mRNAs from rice undergoing resistant or susceptible responses. A collection of rice deletion mutants (approx. 40 000) has been screened for many characters, including increased susceptibility or resistance in rice to Magnaporthe grisea and Xanthomonas oryzae pv. oryzae. Using these resources, we are developing an efficient screen to detect mutants with deletions in DR genes. Arrays of DR genes will be used to create gene expression profiles of interesting mutants. Successful application of the mutant screen will have broad utility in identifying mutants deleted in candidate genes involved in other metabolic pathways. Breeding for nutritional characteristics in cereals Robin D. Graham and Glenn Gregorio Extensive genetic variation within large species such as the major cereals can be confidently expected for any new trait of interest. This has now been extensively demonstrated for the nutrient content of cereal grains that is of interest under deficient conditions both to human nutritionists and to cereal agronomists. As cereals are eaten in large quantity by practically everyone, they are the ideal vehicles for changing the balance of nutrient intake of the whole human population�and doing precisely this appears to be necessary as the World Health Organization has identified deficient micronutrient intake in well over half of all people globally, notably women and children. Of major concern are iron, zinc, calcium and vitamin A related carotenoids. Our results show that for any staple so far studied, the intake of iron, calcium and zinc from cereals can be doubled, and the content/intake of essential carotenoids can be increased by much greater factors. That greater intake results in greater absorption and measurable health benefits is quite difficult to prove to rigid scientific standards but is currently being assessed in various ways. This question of bioavailability is all that impedes implementation in breeding programs. A review of the literature has revealed synergies in aborption and function among these essential nutrients that strongly suggests that the degree of enhancement already possible through naturally occurring genetic variation is all that is necessary to redress the problem of these three nutrient deficiencies in modern diets. Introducing the complete beta-carotene (pro-vitamin A) biosynthesis pathway into rice endosperm by genetic engineering Peter Beyer and Ingo Potrykus* Vitamin A deficiency represents a serious health problem in many countries living on rice as the major staple-food. This is because rice, in its milled form, is free of pro-vitamin A (beta-carotene). In an approach to overcome this problem, we carried out work to introduce pro-vitamin A biosynthesis into rice endosperm by genetic engineering. We used cDNAs from Narcissus pseudonarcissus coding for the carotenogenic enzymes phytoene synthase and lycopene cyclase, both placed under the control of the rice glutelin (Gt1)-promoter, as well as a bacterial (Erwinia) gene coding for phytoene desaturase under 35S promoter control, to transform rice (TP 309). We obtained T0 seeds exhibiting a clearly visible yellow colour due to carotenoid accumulation. Among these, pro-vitamin A was predominant, followed by the xanthophylls lutein and zeaxanthin, as revealed by HPLC analyses. This resulted in a carotenoid complement that is qualitatively not dissimilar to the one observed in green leaves. To have the full carotenoid pattern, including pro-vitamin A and the xanthophylls, may be nutritionally more advantageous, considering the role of zeaxanthin and lutein in preventing macula degeneration of the eye. The appearance of the xanthophylls (and of beta-carotene in some of the experiments) was unexpected, since our transformation did not comprise the corresponding hydroxylase (lycopene cyclase). Our preliminary evidence suggests that later enzymes in the carotenoid biosynthetic pathway are induced by products derived from the transformation. The results obtained will be discussed and details will be given. Developing transgenic grains with improved oils, proteins and carbohydrates Barbara J Mazur DuPont has developed cereals and oilseeds with improved proteins, carbohydrates, and oils for food, feed and industrial applications. Products which have been or will be introduced include corn and soybeans with increased oil content, improved oil composition, increased amino acid content, altered protein content and functional qualities, altered starch composition, reduced oligosaccaride content, increased sucrose content, and combinations of these traits. These products have been developed using both mutation breeding and molecular biology-based transgenic approaches. We have also worked on improving the underlying technologies in order to accelerate product introductions. Gene discovery has been expedited through a genomics program that now has a database of more than 1.5 million sequences from a variety of plants, insects, and microbes. Plant cell transformation for elite lines of crop species is being addressed through production laboratories with high throughput processes and through technology improvements. High-throughput, rapid and small-scale assays for biochemical parameters are used to identify plants carrying traits of interest. Small-scale functionality analyses, in which grains are broken down into their component parts and assayed for functional properties, indicate which seeds carry a trait of commercial value. Finally, a number of DNA marker systems are being used to accelerate trait introgression timelines. OTHER CONTRIBUTORS John Bennett Howarth Bouis Malcolm Elliott Mahabub Hossain Gurdev Khush (chair) Hei Leung Zhi-Kang Li David Nevill Tony Pryor John Salmeron Guo-Liang Wang Back to issue 2
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