Tuesday, March 22, 2011

Ralf Welsch

“Biofortification” of crop plants describes the idea of providing the micronutrients through the staple crop plant’s own biosynthetic (vitamins) or physiological (minerals) capacity1. This can be accomplished by conventional
classical breeding, when the desired micronutrient-rich germplasm is available, allowing the transfer of such nutritional traits into agriculturally relevant cultivars. In the absence of sufficient genetic variability or when plant breeding is ineffective, the introduction of the desired trait by genetic engineering is required. An important scientific prerequisite for the application of genetic engineering is inter alia a toolbox of identified
genes. The term “Nutritional Genomics” has been coined to describe a discipline of modern plant biology that focuses on the molecular elucidation of the biochemical pathways or physiological processes involved in micronutrient accumulation2.

Biofortification as a necessary vitamin A deficiency intervention

Vitamin A malnutrition is widespread in the tropics, leading to irreversible blindness and severely exacerbating infectious diseases due to its essential role in the immune response. According to the WHO, an estimated 127 million preschool children are affected by vitamin A deficiency, with 250 000 – 500 000 children becoming blind every year, half of whom die within a year (WHO database of vitamin A deficiency; http://www.who.int/vmnis/vitamina/data/en/index.html). Vitamin A denotes a group of compounds formed by the cleavage of provitamin A carotenoids, i.e., carotenoids containing at least one unsubstituted β-ionone ring, such as β- or α-carotene. Prevalence of Vitamin A deficiency among poor people with diets primarily based on rice prompted the development of Golden Rice, which is a rice cultivar that accumulates provitamin A carotenoids in the endosperm, the edible part of the rice grain3,4. The absence of variability for the trait (no germplasm accession is known that develops yellow endosperm) required the use of genetic engineering for biofortification.
Cassava is another important staple crop, cited as the fifth most important crop worldwide. Its importance is even higher in arid areas, such as sub-Saharan Africa. Roots of commercial cassava cultivars are rich in starch, but low in proteins and micronutrients, including provitamin A carotenoids; thus biofortified cultivars with elevated levels of provitamin A are desirable. In contrast to rice, some yellow-rooted cassava varieties do exist, and thus, breeding efforts have yielded a three-fold increase in provitamin A content. However, cassava is vegetatively propagated and breeding is very tedious because of long breeding cycles. Moreover the very complex genetics of cassava renders varietal recovery extremely difficult. Thus, the identification of genes crucial for improvements of the provitamin A content in cassava storage roots can greatly assist in accelerating the development of provitamin A-rich cassava both by breeding and genetic engineering.
We therefore focused on the carotenoid biosynthesis pathway in cassava. We took advantage of existing cassava varieties differing in root carotenoid accumulation and found an allelic polymorphism in the gene coding
for the rate-limiting enzyme of carotenoid biosynthesis. This polymorphism was found to determine root color differences caused by varying accumulations of provitamin A carotenoids5.

SNPs within cassava phytoene synthase 2

Three cassava cultivars accu-mulating different carotenoid amounts in their storage roots were used in the study: a white-colored CM3306-4, a yellow-colored MBRA253, which contained about ten-fold more carotenoids
than white-colored roots, and an intermediately-colored cultivar CM2772-3. The RNA expression levels of major carotenoid biosynthesis genes in all three cultivars were very similar; thus differential gene expression at this level cannot explain the phenotypic differences. These analyses included the enzyme phytoene synthase (PSY), which represents the rate-limiting step in carotenoid biosynthesis in many systems. For instance, high carotenoid levels in tomato fruits correlate with high transcript levels of one PSY orthologue. Cassava contains three PSY genes in its genome, but transcripts from all three PSY genes showed similar levels in all three cultivars.
However, we observed that the protein-coding sequence of one orthologue, PSY2, was not identical in all three varieties—in total, three single nucleotide polymorphisms (SNP) were determined. While one SNP was synonymous, the two remaining SNPs were present only in the intermediately- and yellow-colored cultivars (Fig. 1A). These SNPs introduced changes in the amino acid sequence, potentially impacting the enzymatic activity.
To confirm this hypothesis, we took advantage of a crossing population derived from a white-rooted and a yellow-rooted cassava cultivar (white: MMAL66, yellow: MBRA1a; Fig. 1B). The PSY2 gene sequence revealed that only one of the two non-synonymous SNPs segregated with the yellow-rooted individuals in the offspring: the selfed offspring of white-rooted F1 individuals remained white and were all homozygous for one PSY2 allele. In contrast, the yellow root color in selfed yellow F1 individuals segregated and correlated with the presence of the second allele. Suggesting dominance, homozygosity for this allele was not required for producing the yellow root phenotype.

A single amino acid change affects PSY

Based on the PSY2 amino acid sequence from white-rooted cassava cultivars, SNP within the PSY2 allele from yellow-colored cassava varieties prompts a change from alanine (PSY2A191) to aspartate (PSY2D191) at position 191 (Fig. 1C). Using heterologous expression in yeast cells that co-expressed the substrate-delivering enzyme geranylgeranyl diphosphate synthase, the impact of the amino acid change on enzymatic activity was confirmed.
Yeasts expressing the PSY2 version from yellow-colored cassava cultivars (PSY2D191) formed twice as much phytoene as those transformed with the version from white-colored cultivars (PSY2A191). Moreover, the introduction of the corresponding amino acid exchange in PSY enzymes from other species, such as Arabidopsis, daffodil, and maize, led to increased enzymatic activity, as tested with an E. coli based expression system.
Allelic variants of PSY that influence carotenoid formation have been reported previously. For instance, allelic
PSY1 variants of durum wheat have been associated with high endosperm carotenoid content, but none of these mutations were in a position equivalent to the one reported here6. A systematic assessment of the impacts of these amino acid changes on PSY activity could be used to select favorable alleles using marker-assisted selection.
Moreover, the synergistic effects of activity-increasing amino acid changes identified in different PSY variants might appear when merging them within one polypeptide. Similarly, using these test systems, unbiased
approaches with recombinant PSY versions, generated, for example, by directed evolution, might further lead to activity-optimized PSY versions suitable to increase the metabolic flux in the carotenoid biosynthetic pathway.
Rate-limitation of phytoene synthase is given also in cassava roots.
The determination that a single amino acid difference in PSY2 results in higher enzymatic activity and thus in higher total carotenoid levels in cassava roots underscores the rate-limiting function of this enzyme. In transformation approaches, increased metabolic flux was achieved through overexpression, i.e., through increased PSY protein amounts. This was done by overexpressing a bacterial PSY, CrtB, in cassava storage roots. In fact, transgenic lines accumulated more than 30 times higher total carotenoid levels than wild-type roots (see Fig. 2). Interestingly, in the strongest line, non-colored carotene intermediates represented about 50%. It cannot be excluded that this results from the properties of the (bacterial) PSY used and that a more effective phytoene metabolization is achievable upon overexpression of a plant-derived
version, including the cassava PSY2 allele discovered.
Alternatively, the release of one rate limitation within the pathway might have led to the uncovering of a subsequent one. Further work is needed to distinguish between these possibilities.
Thus, our first approach towards genetically engineered provitamin A-rich cassava must be regarded as a starting point to further improve the provitamin A biosynthetic potential.

Usefulness of the findings

The identification of the role of the cassava PSY2 allele provides a tool for marker-assisted breeding to facilitate the development of provitamin A-dense cassava storage roots. Given the constraints in cassava breeding, the application of transgenic technologies to integrate desired traits into farmer-preferred cultivars is highly  attractive, taking advantage of storage root-specific promoters recently identified7,8. An additional benefit of provitamin A-accumulating cassava roots is related to a major constraint in the storing and marketing of cassava due to rapid postharvest physiological deterioration of the roots (PPD)9. PPD, observed as a blackish vascular streaking accompanied by secondary microbial deterioration, can occur only hours after harvest and renders the roots unpalatable. Recent evidence suggests that the presence of provitamin A causes a reduction or delay of PPD in cassava10. This would be a very desirable trait because cassava is mostly grown in environments frequently characterized by large distances to the processing centers and deficient transport infrastructure. Further research is underway to clarify the role of carotenoids in this process.

A non-synonymous SNP was identified in the protein-coding region of PSY2 from yellow-colored cassava cultivars. Root transverse sections and sequencing chromatograms of the corresponding PSY2 region in white (CM3306-4) and yellow-colored (MBRA 253) cassava cultivars are shown in A. This SNP cosegregated with yellow root color in a breeding pedigree derived from a cross between the white-rooted cultivar MMAL66 and the yellow-rooted MBRA1A (B). The resulting amino acid change results in a change from alanine in white-rooted (Cassava#2-W) to aspartate in yellow-colored cultivars (Cassava#2-Y; indicated in red in C; PSY sequences from tomato, daffodil, Arabidopsis and maize are shown for comparison).

References
1. Mayer JE, Pfeiffer WH & Beyer P. Biofortified crops to alleviate micronutrient malnutrition. Curr Opin Plant Biol 11, 166-70 (2008).
2. DellaPenna D. Nutritional Genomics: Manipulating plant micronutrients to improve human health. Science 285, 375-9 (1999).
3. Ye X, Al-Babili S et al. Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287, 303-5 (2000).
4. Paine JA et al. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature biotechnology 23, 482-7 (2005).
5. Welsch R, Arango J et al. Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene. Plant Cell 22: 3348-56 (2010).
6. Singh A et al. Allelic variation at Psy1-A1 and association with yellow pigment in durum wheat grain. Theor Appl Gen 118, 1539-48 (2009).
7. Beltrán J et al. Expression pattern conferred by a glutamic acid-rich protein gene promoter in field-grown transgenic cassava (Manihot esculenta Crantz). Planta 231, 1413-24 (2010).
8. Arango J et al. Putative storage root specific promoters from cassava and yam: cloning and evaluation in transgenic carrots as a model system. Plant Cell Rep 29, 651-9 (2010).
9. Beeching JR et al. Wound and defense responses in cassava as related to post-harvest physiological deterioration. Recent Adv Phytochem 32, 231-48, (1998).
10. Sánchez T et al. Reduction or delay of post-harvest physiological deterioration in cassava roots with higher carotenoid content, J Sci Food Agric 86, 634-39 (2006).