Joyce Van Eck

The Cauliflower Or Gene Encodes a DnaJ Cysteine-Rich Domain-Containing Protein That Mediates High Levels of β-Carotene Accumulation

Despite recent progress in our understanding of carotenogenesis in plants, the mechanisms that govern overall carotenoid accumulation remain largely unknown. The Orange (Or) gene mutation in cauliflower (Brassica oleracea var botrytis) confers the accumulation of high levels of β-carotene in various tissues normally devoid of carotenoids. Using positional cloning, we isolated the gene representing Or and verified it by functional complementation in wild-type cauliflower. Or encodes a plastid-associated protein containing a DnaJ Cys-rich domain. The Or gene mutation is due to the insertion of a long terminal repeat retrotransposon in the Or allele. Or appears to be plant specific and is highly conserved among divergent plant species. Analyses of the gene, the gene product, and the cytological effects of the Or transgene suggest that the functional role of Or is associated with a cellular process that triggers the differentiation of proplastids or other noncolored plastids into chromoplasts for carotenoid accumulation. Moreover, we demonstrate that Or can be used as a novel genetic tool to induce carotenoid accumulation in a major staple food crop. We show here that controlling the formation of chromoplasts is an important mechanism by which carotenoid accumulation is regulated in plants.

A new class of regulatory genes underlying the cause of pear-shaped tomato fruit

A common, recurring theme in domesticated plants is the occurrence of pear-shaped fruit. A major quantitative trait locus (termed ovate) controlling the transition from round to pear-shaped fruit has been cloned from tomato. OVATE is expressed early in flower and fruit development and encodes a previously uncharacterized, hydrophilic protein with a putative bipartite nuclear localization signal, Von Willebrand factor type C domains, and an ≈70-aa C-terminal domain conserved in tomato, Arabidopsis, and rice. A single mutation, leading to a premature stop codon, causes the transition of tomato fruit from round- to pear-shaped. Moreover, ectopic, transgenic expression of OVATE unevenly reduces the size of floral organs and leaflets, suggesting that OVATE represents a previously uncharacterized class of negative regulatory proteins important in plant development.

Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System

The CRISPR/Cas9 system is highly efficient at generating targeted mutations in stable transgenic tomato plants, and homozygous deletions of a desired size can be created in the first generation.

Setaria viridis: A Model for C4 Photosynthesis

C4 photosynthesis drives productivity in several major food crops and bioenergy grasses, including maize (Zea mays), sugarcane (Saccharum officinarum), sorghum (Sorghum bicolor), Miscanthus x giganteus, and switchgrass (Panicum virgatum). Gains in productivity associated with C4 photosynthesis include improved water and nitrogen use efficiencies. Thus, engineering C4 traits into C3 crops is an attractive target for crop improvement. However, the lack of a small, rapid cycling genetic model system to study C4 photosynthesis has limited progress in dissecting the regulatory networks underlying the C4 syndrome. Setaria viridis is a member of the Panicoideae clade and is a close relative of several major feed, fuel, and bioenergy grasses. It is a true diploid with a relatively small genome of ~510 Mb. Its short stature, simple growth requirements, and rapid life cycle will greatly facilitate genetic studies of the C4 grasses. Importantly, S. viridis uses an NADP-malic enzyme subtype C4 photosynthetic system to fix carbon and therefore is a potentially powerful model system for dissecting C4 photosynthesis. Here, we summarize some of the recent advances that promise greatly to accelerate the use of S. viridis as a genetic system. These include our recent successful efforts at regenerating plants from seed callus, establishing a transient transformation system, and developing stable transformation.

Effect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers

Transgenic plants have facilitated our understanding of the functional roles of genes and the metabolic processes affected in plants. Recently, the Or gene was isolated from an orange cauliflower mutant and it was shown that the Or gene could serve as a novel genetic tool to enrich carotenoid content in transgenic potato tubers. An in-depth characterization of these Or transgenic lines is presented here. It was found that the Or transgene may facilitate the identification of potential rate-limiting step(s) of the carotenoid biosynthetic pathway. The Or transgenic tubers accumulated not only increased levels of carotenoids that normally are present in the controls, but also three additional metabolite intermediates of phytoene, phytofluene, and zeta-carotene, indicating that the desaturation steps became limiting following the expression of the Or transgene. Moreover, we observed that long-term cold storage greatly enhanced carotenoid content in the Or transgenic tubers to a level of 10-fold over controls. Expression of the Or transgene in the transgenic plants caused no dramatic changes in the transcript levels of the endogenous carotenoid biosynthetic genes, which is in agreement with the Or gene not directly controlling carotenoid biosynthesis. Microscope analysis revealed that the Or transgene conferred the formation of chromoplasts containing carotenoid sequestering structures in a heterologous system. Such structures were not observed in tubers of potato cultivars that accumulate high levels of carotenoids. Collectively, these results provide direct evidence demonstrating that the Or gene indeed controls chromoplast differentiation and that regulation of chromoplast formation can have a profound effect on carotenoid accumulation in plants.

The Tomato Sequencing Project, the First Cornerstone of the International Solanaceae Project (SOL)

The genome of tomato (Solanum lycopersicum) is being sequenced by an international consortium of 10 countries (Korea, China, the United Kingdom, India, The Netherlands, France, Japan, Spain, Italy and the United States) as part of a larger initiative called the ‘International Solanaceae Genome Project (SOL): Systems Approach to Diversity and Adaptation’. The goal of this grassroots initiative, launched in November 2003, is to establish a network of information, resources and scientists to ultimately tackle two of the most significant questions in plant biology and agriculture: (1) How can a common set of genes/proteins give rise to a wide range of morphologically and ecologically distinct organisms that occupy our planet? (2) How can a deeper understanding of the genetic basis of plant diversity be harnessed to better meet the needs of society in an environmentally friendly and sustainable manner? The Solanaceae and closely related species such as coffee, which are included in the scope of the SOL project, are ideally suited to address both of these questions. The first step of the SOL project is to use an ordered BAC approach to generate a high quality sequence for the euchromatic portions of the tomato as a reference for the Solanaceae. Due to the high level of macro and micro-synteny in the Solanaceae the BAC-by-BAC tomato sequence will form the framework for shotgun sequencing of other species. The starting point for sequencing the genome is BACs anchored to the genetic map by overgo hybridization and AFLP technology. The overgos are derived from approximately 1500 markers from the tomato high density F2-2000 genetic map (http://sgn.cornell.edu/). These seed BACs will be used as anchors from which to radiate the tiling path using BAC end sequence data. Annotation will be performed according to SOL project guidelines. All the information generated under the SOL umbrella will be made available in a comprehensive website. The information will be interlinked with the ultimate goal that the comparative biology of the Solanaceae—and beyond—achieves a context that will facilitate a systems biology approach.

A cascade of arabinosyltransferases controls shoot meristem size in tomato

Shoot meristems of plants are composed of stem cells that are continuously replenished through a classical feedback circuit involving the homeobox WUSCHEL (WUS) gene and the CLAVATA (CLV) gene signaling pathway. In CLV signaling, the CLV1 receptor complex is bound by CLV3, a secreted peptide modified with sugars. However, the pathway responsible for modifying CLV3 and its relevance for CLV signaling are unknown. Here we show that tomato inflorescence branching mutants with extra flower and fruit organs due to enlarged meristems are defective in arabinosyltransferase genes. The most extreme mutant is disrupted in a hydroxyproline O-arabinosyltransferase and can be rescued with arabinosylated CLV3. Weaker mutants are defective in arabinosyltransferases that extend arabinose chains, indicating that CLV3 must be fully arabinosylated to maintain meristem size. Finally, we show that a mutation in CLV3 increased fruit size during domestication. Our findings uncover a new layer of complexity in the control of plant stem cell proliferation.

Advancing Crop Transformation in the Era of Genome Editing

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized.

Eggplant (Solanum melongena L.).

Eggplant is an economically important vegetable crop in Asia and Africa, and although it is grown in Europe and the United States, it does not account for a significant percentage of agricultural production. It is susceptible to a number of pathogens and insects, with bacterial and fungal wilts being the most devastating. Attempts to improve resistance through introgression of traits from wild relatives have had limited success owing to sexual incompatibilities. Therefore, a crop improvement approach that combines both conventional breeding and biotechnological techniques would be beneficial. This chapter describes an Agrobacterium-mediated transformation protocol for eggplant based on inoculation of seedling explants (cotyledons and hypocotyls) and leaves. We have used this protocol to recover transformants from two different types of eggplant, a Solanum melongena L. breeding line, and S. melongena L. var. Black Eggplant. The selectable marker gene used was neomycin phosphotransferase II (nptII) and the selection agent was kanamycin. In vitro grown transformants acclimated readily to greenhouse conditions.

Variation in the flowering gene SELF PRUNING 5G promotes day-neutrality and early yield in tomato

Plants evolved so that their flowering is triggered by seasonal changes in day length. However, day-length sensitivity in crops limits their geographical range of cultivation, and thus modification of the photoperiod response was critical for their domestication. Here we show that loss of day-length-sensitive flowering in tomato was driven by the florigen paralog and flowering repressor SELF-PRUNING 5G (SP5G). SP5G expression is induced to high levels during long days in wild species, but not in cultivated tomato because of cis-regulatory variation. CRISPR/Cas9-engineered mutations in SP5G cause rapid flowering and enhance the compact determinate growth habit of field tomatoes, resulting in a quick burst of flower production that translates to an early yield. Our findings suggest that pre-existing variation in SP5G facilitated the expansion of cultivated tomato beyond its origin near the equator in South America, and they provide a compelling demonstration of the power of gene editing to rapidly improve yield traits in crop breeding.