Nutritional and safety assessments of foods and feeds nutritionally improved through biotechnology.

Maize is an important staple of human diets and animal feed. Improving the nutritional profile for bothof these purposes is a desirable research focus and agricultural endpoint. Many mechanisms have been used toimprove maize protein and oil content, including selective breeding to introgress desirable traits (Quality ProteinMaize), introducing single natural or synthetic genes expressing desired traits, changing signal sequences (highmethionine maize), and modifying metabolic pathways (high-lysine maize, see Chapter 6). A novel approach wasused to indirectly increase protein and oil content (Young and others 2004). Maize produces unisexual florets.Within the maize spikelet, the meristem gives rise to an upper and lower floret and male (tassel)and female (ear)-specific florets are borne on separate inflorescences. The lower floret of each ear spikelet is aborted early in its devel-opment, leaving the upper floret to mature as the only female floret. Expression of a bacterial cytokinin-synthesiz-ing isopentenyl transferase (IPT) enzyme, under the control of the Arabidopsis senescence-inducible promoter SAG12 (senescence associated gene), blocked the abortion of the lower floret and resulted in 2 functional florets perspikelet. The pistil in each floret was fertile, but the spikelet produced just 1 kernel composed of a fused endospermwith 2 viable embryos. The 2 embryos were genetically distinct, indicating that they had arisen from independentfertilization events. The embryo contains most of the protein and oil in the kernel and kernels that contained 2embryos have more protein than conventional maize. The presence of 2 embryos in a normal-sized kernel leads todisplacement of endosperm growth, resulting in kernels with an increased ratio of embryo to endosperm content.The end result is maize with more protein and oil and less carbohydrate (Young and others 2004). peculiarity of certain plants that has evolved as a reproductivestrategy to achieve such an endpoint for two of the principalcomponents in maize, protein and oil content. 3.2 Experimental ProcedureTo put the current work in context from a nutritional and safe-ty assessment perspective, it is important to provide some back-ground on kernel development in maize. Unlike the majority ofplant species, maize produces unisexual florets that have onlyeither male (stamen) or female (pistil) parts, but both male andfemale flowers appear on the same plant (monoecious). Formonoecious species, the sex determination process by whichsexual organs form in each floret is rather complex. Within themaize spikelet, the meristem gives rise to an upper and a lowerfloret, and male (tassel)and female (ear)-specific florets areborne on separate inflorescences. The male stamen (tassel) isresponsible for producing pollen. The female pistil (ear) bearsthe ovaries that give rise to kernels after pollination. Althoughinitially bisexual, floret unisexuality is established throughselective organ elimination and abortion of specific floretorgans. Female initials are eliminated from florets in the tasselinflorescence, resulting in male florets; conversely, male initialsare eliminated from florets in the ear inflorescence, resulting infemale florets. In addition, the lower floret of each ear spikeletis aborted early in its development, leaving the upper floret tomature as the only female floret. Arrest of pistil development inthe lower floret is followed by degeneration, which initiates inthe pistil primordium (precursor cells).As with many organisms, this selective loss is achievedthrough apoptosis or programmed cell death (PCD), a funda-mental process that cuts across the boundaries of many scientificdisciplines. In the context of maize reproduction, it is interestingthat the Greek term apoptosis traces its roots not to a medicalphenomenon, but rather to the process of a petal falling from aflower or the loss of a leaf from a tree. As with other apoptoticapplications in plant development, the process results in dis-carding tissues or organs that are no longer required, modifyingexisting organs, or adapting to environmental changes.Induction of PCD is a fine balance between the withdrawal ofpositive signals; that is, signals needed for continued survival,and the receipt of negative signals. The continued survival ofmost cells requires that they receive continuous stimulationfrom other cells.There are several naturally occurring maize mutations calledtasselseed (for example, ts1 and ts2) that result in female flow-ers developing on the male tassel. The ts2 mutation has beencloned and encodes a short-chain alcohol dehydrogenase.Normal expression of ts2 in the pistil primordium of male flow-ers induces apoptosis and leads to pistil abortion and male tas-sel development. Both genes are suppressed by silkless1 (sk1).Loss of sk1 leads to the abortion of both upper and lower floretpistil in ear spikelets. In contrast, loss of ts1 or ts2 functionresults in the sexual maturation of pistils in all florets, tassels,and upper and lower florets of each ear spikelet, leading to acomplete female takeover. Expression of ts2 seems to switch onPCD because it coincides with the loss of nuclei in the pistil pri-mordium. The presence of Sk1 basically eliminates ts2 activityin the upper floret pistil by blocking PCD, although ts2 isexpressed in all pistil primordial cells (Calderon-Urrea andDellaporta 1999). Mutations at 2 other loci (pi1/pi2) differential-ly suppress pistil abortion in the lower ear floret, but not in thetassel spikelets (Veit and others 1993). The fact that this demon-strates a mechanism (even if the actual process is unknown)whereby one can rescue the lower floret pistil, while not affect-ing pistil abortion in tassel spikelets (thus not affecting male fer-tility), makes for a potential intervention point that was takenadvantage of with some unexpected payoffs from a nutritionalperspective (Young and others 2004). Intervention was achievedby the judicious use of plant growth regulators (PGRs) ratherthan direct manipulation of gene activity, per se.It had already been demonstrated in tobacco leaves thatcytokinin plays a key role in regulating entry into a senescenceprogram. Cytokinins are adenine-derived compounds that pro-mote cell division and have other similar functions to kinetin.Gan and Amasino (1995) developed a system based oncytokinins to study leaf senescence because it has importantagricultural implications. For example, senescence may limityield in certain crops, and it also contributes to the postharvestloss of vegetable crops. Therefore, studying leaf senescencecould lead to ways of manipulating senescence for agriculturalapplications. Gan and Amasino (1995) developed a construct tostudy the effect of cytokinin by placing the Agrobacteriumcytokinin-synthesizing IPT gene under the control of theArabidopsis senescence-inducible promoter from the cysteineCRFSFS: Comprehensive Reviews in Food Science and Food Safety 76 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 7, 2008Nutritional values of corn grain (wt per 100 g)