Cereal crops that can fix their own N directly from the atmosphere have long been a dream of scientists. Two approaches have been explored: to introduce nitrogenase directly into the plant so that it can fix N2directly; and, more recently, to manipulate the cereal plant so that it can nodulate with rhizobia.
Introducing nitrogenase into cereal crops
The principal target for introduction of nitrogenase to cereals is the chloroplast. It is now widely accepted that chloroplasts originated as symbiotic bacteria, and genes in chloroplasts are expressed in a similar way as in bacteria (Merrick and Dixon, 1984).
The major problem envisaged for active N2-fixation in chloroplasts is protection of nitrogenase from molecular oxygen produced during photosynthesis. As described in Chapter 3, the unicellular cyanobacteriumGloeothecesynthesizes nitrogenase in the dark but is then able to continue to fix N2when returned to the light. By analogy it has been proposed that plants could be engineered to be nocturnal N2-fixers, but extra challenges remain. Such a plant system must ensure the energy supply for nitrogenase throughout the night, and ensure that nitrogenase could be protected to avoid the need for dailyde novosynthesis of the enzyme.
One of the many nitrogenase genes,nifH, has been inserted into the chlorophyll genome of the green algaChlamydomonas, which is being used as a model system for higher plants (Dixonet al., 2000). Although this gene appears to be expressed in Chlamydomonas, there has been surprisingly little advance in this field since the early 1980s, presumably due to a lack of targeted funding for the research. Dixonet al.
(2000) suggested that the oxygen-tolerant nitrogenase recently described in S.
thermoautotrophicus(Ribbeet al., 1997) (Chapter 3) may be a promising alternative model for overcoming the oxygen problem in chloroplasts. Unfortunately, a set of different biochemical problems are envisaged in this case, most notably the genera- tion of superoxide that the eukaryotic cell may not tolerate. Whichever approach is pursued, a functioning eukaryotic nitrogenase will require the additional machinery to supply reductant and electrons in adequate amounts, which may be much more problematic than engineering plants to manufacture nitrogenase.
Nodulating cereals
Substantial research on achievement of N2-fixing nodules in cereal crops was stimulated by the observation that ‘nodular structures’ could be induced on roots of rice andBrassica napus(Al-Mallahet al., 1990a,b; Cockinget al., 1990; Jinget al., 1990). Cocking’s group induced nodules inBrassica and rice by treating the roots with cellulase and pectolyase in the presence of polyethylene glycol (to prevent lysing of cells when the cell wall was degraded). Surprisingly poor microscopic evidence was presented for invasion of the plant tissues by bacteria, and insignificant amounts of ethylene production were reported (see Chapter 4 for a discussion of the pitfalls of this method). A follow-up of the reports of Jing, Li and colleagues (Jinget al., 1990;
Liet al., 1991) revealed that the ‘nodules’ on rice appeared to be formed as a result of fungal infections, or were short lateral root meristems, and attempts to reisolate rhizobia from these ‘paranodules’ failed (de Bruijnet al., 1995).
Particular attention is being devoted to exploring the prospects for nodulation and N2-fixation in rice, as the potential benefits are immense (Khush and Bennett,
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1992; Ladhaet al., 1997; Ladha and Reddy, 2000). This ‘frontier project’ aims to increase N2-fixation by associated and endophytic bacteria, and to enhance the efficiency of general N metabolism in addition to exploring prospects for formation of N2-fixing nodules in rice. Close examination reveals that ‘nodular structures’
reported on rice roots have a central vasculature and are, in fact, stubby lateral roots.
Reddy et al. (1997), in a study with excellent attention to detail, examined the predisposition of rice to infection by rhizobia. Although rhizobia were able to gain entry into rice roots, the association was non-specific, with only passive involvement of the plant and no evidence for recognition between the bacteria and the plant roots.
A suite of other characters in rice differ from the symbiosis between legumes and rhizobia: rice did not induce rhizobialnodgenes; no true nodule (or induction of lateral roots) occurred; there was no root hair attachment or cellulose microfibril development; bacteria were present only in intracellular spaces or in lysing cells; and a schlerenchymatous layer of cells denied the rhizobia admission to the deeper layers of the root cortex.
On the other hand, roots of rice and most other cereal crops are infected by mycorrhizas, and this may provide some clues to establishment of closer associations with rhizobia. Homologues of nodulin genes, which are expressed specifically during nodule development in legumes, are present in rice and other grasses (Reddyet al., 1999). In rice, one nodulin gene appears to be expressed in the early development of vascular bundles (Kouchiet al., 1999). Current thinking and future approaches to achieving nodulation and N2-fixation in rice are discussed in detail in Ladha and Reddy (2000).
Conclusions
Knowledge of the occurrence and biology of heterotrophic N2-fixers has now increased enormously, but we still, shamefully, lack good methods for accurately quantifying the amounts of N2 fixed. The case for an agriculturally ‘significant’
contribution of fixed N remains to be proved, but in natural grasslands, over long time-periods, the small inputs from free-living or associative N2-fixation are undoubtedly valuable (Giller and Day, 1985).
Given the abundant supply of sucrose and the presence in its tissues of A. diazotrophicus, which can utilize sucrose to fix N2(Gilliset al., 1989), sugarcane in particular seems a very good candidate for supporting significant amounts of heterotrophic N2-fixation (Boddey, 1995). To date there is little evidence to indicate that endophytic N2-fixation is more important than N2-fixation in the rhizosphere.
Whilst proof of N2-fixation by endophytes can be gained by incubating plants inoculated with N2-fixing bacteria ornif-mutants in the presence of15N2gas, or by assessing ability of such inoculated plants to grow in N-free media (Chapter 4), this will not help in understanding what happens in the field. Long-term N balance studies, in which all sources of N are carefully monitored and controlled over periods
of 10 to 20 years, are probably the only way of understanding whether N2-fixation with sugarcane can sustain production. Although research has been initiated to explore the transfer of the ability to fix N2to cereals, this still remains a fairly distant prospect.
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Cyanobacteria andAzolla Chapter 7