Transgenic biofortification of the starchy staple cassava (Manihot esculenta) generates a novel sink for protein

Abstract

Although calorie dense, the starchy, tuberous roots of cassava provide the lowest sources of dietary protein within the major staple food crops (Manihot esculenta Crantz). (Montagnac JA, Davis CR, Tanumihardjo SA. (2009) Compr Rev Food Sci Food Saf 8:181-194). Cassava was genetically modified to express zeolin, a nutritionally balanced storage protein under control of the patatin promoter. Transgenic plants accumulated zeolin within de novo protein bodies localized within the root storage tissues, resulting in total protein levels of 12.5% dry weight within this tissue, a fourfold increase compared to non-transgenic controls. No significant differences were seen for morphological or agronomic characteristics of transgenic and wild type plants in the greenhouse and field trials, but relative to controls, levels of cyanogenic compounds were reduced by up to 55% in both leaf and root tissues of transgenic plants. Data described here represent a proof of concept towards the potential transformation of cassava from a starchy staple, devoid of storage protein, to one capable of supplying inexpensive, plant-based proteins for food, feed and industrial applications.

Figure 1. Protein accumulation in leaves and peeled storage roots of transgenic cassava expressing zeolin under control of the patatin and 35S promoters.

A) Protein content in storage roots as quantified by Bradford assay after 7 months of growth in pots in the greenhouse. Non-transgenic cassava cv. 60444 (dark green bar) and transgenic plant line with empty gene vector only (pCambia2300) (light green bar) show total protein content of less than 3% dw, while all seven transgenic lines expressing zeolin under control of the patatin promoter (blue bars) accumulate total protein at 9.5% to 10.3% dw. Storage roots from plants expressing zeolin under control of the 35S promoter (orange bars) do not accumulate protein at levels above controls. B) Accumulation of total protein in peeled storage roots of pot grown transgenic plants expressing zeolin under control of the patatin promoter with time, as assessed by Bradford assay. Total protein content of non-transgenic cassava cv. 60444 (green bar) shows no increase over a 7 month cultivation period in the greenhouse, while the three transgenic events studied accumulated protein at slightly differing rates, but all reached approximately 10% dw by the end of the 7 month period. C) SDS-PAGE of crude protein extracted from leaves and roots of in vitro transgenic cassava lines expressing the zeolin transgene. Forty micrograms of protein extract were loaded as follows: lane 1: protein ladder; lanes 2 and 3: non-transgenic cassava cv. 60444 leaves and roots respectively; lanes 4 and 5: leaves and roots of transgenic cassava line transformed with pILTAB601 in which 35S drives expression of zeolin; and lanes 6 and 7: leaves and roots transgenic cassava line transformed with pILTAB600 in which the patatin promoter drives expression of zeolin. Presence of distinct band at 65 kDa in transgenic plants, but not in the control, indicates accumulation of zeolin in leaves of plants transgenic for 35S-zeolin and roots of plants transgenic for patatin-zeolin, but not vice versa. D) Western blot analysis of leaves (L) and peeled storage roots (R) harvested from greenhouse soil beds at 7 months of age. Total protein was isolated, 100 µg loaded in each lane and detected with specific anti-zein antibodies. Presence of zeolin protein was detected in all tissues, but accumulated at 3.5 times greater amounts in roots when expressed under the control of patatin versus the 35S promoter with the reverse pattern seen in leaves. E) Immuno-printing of 50–100 µm thick sections of cassava storage roots from transgenic line pILTAB600-25 in which the patatin promoter drives zeolin. Plants were grown in pots in the greenhouse and harvested from one to sevens months of age with non-transgenic cv. 60444 used as a control (top right panel). Presence of zeolin is clearly seen in roots of all ages in the transgenic, but not the control root sections, with significant accumulation in the core (xylem parenchyma) as well as the outer peel layer. Bars = 1 cm.

Figure 2. Characterization of fully-grown zeolin expressing plants.

A) Total protein content of peeled storage roots harvested from transgenic cassava plants expressing zeolin grown in greenhouse soil beds and in the field. Non-transgenic plant 60444 (green bar); plant line transgenic for empty gene vector control (pCambia2300) (light green bar); plants transgenic for patatin promoter-zeolin (pILTAB600; blue bars); plants transgenic for 35S promoter-zeolin (pILTAB601; orange bar) n = 7. Assessment of total protein content by Bradford assay of storage roots shows similar results from both locations with non-transgenic, empty vector control and 35S driven zeolin varying from 2.9 to 3.1% dw, and all three events transgenic for patatin driven zeolin accumulating 3 to 4 times this amount of total protein. Total protein content from roots of field-grown plants was higher in all three lines compared to greenhouse-grown, with maximum levels of total protein reaching 12.3% dw in line pILTAB600-25. B) Evaluation of important agronomic traits of zeolin expressing cassava plants grown for 11 months in the field in Puerto Rico. Non-transgenic cv. 60444 plant line (green bar), plant line transgenic for empty gene vector control (pCambia2300) (light green bar), plants transgenic for patatin promoter driven zeolin transgenic lines (pILTAB600) (blue bars); plants transgenic for 35S promoter driven zeolin (orange bar) n = 12. Transgenic events showed no significant differences compared to controls across traits studied.

Figure 3. Analysis of amino acid and cyanogenic content in cassava tissues transgenic for zeolin.

A) Total amino acid profile of storage roots of non-transgenic cassava cv. 60444 (green bars) and transgenic line pILTAB600-25 (blue bars) in which zeolin is driven by the patatin promoter. Increase in amounts of individual amino acids in transgenic root tissue varied from a four- to ninefold increase compared to the non-transgenic control in a manner that reflects the expect amino acid profile for zeolin. B, C) Levels of cyanogens present in cassava leaf and root tissues of seven-month-old, pot grown plants. All transgenic lines showed a reduction in cyanogen content within leaves and storage roots compared to the non-transgenic control and was apparent in transgenic events in which zeolin was under control of either the patatin (pILTAB600, blue bars) or 35S (pILTAB601, orange bars) promoters. A maximum reduction of 55% was observed in roots of pILTAB600-25, the transgenic event that accumulated the highest levels of total protein within its storage roots.