Regulation of zein synthesis by nitrogen nutrition in cultured endosperms

--C. Balconi, E. Rizzi, M. Motto, F. Salamini and R. Thompson

The maize gene opaque-2 (O2) controls the expression in developing endosperm of the 22kDa zeins and of an abundant cytosolic albumin, termed b-32 (Motto, M et al., Oxford Surv. Plant Mol. Cell. Biol. 6:67-114, 1989). Plants homozygous for the o2 mutation typically show a 50-70% reduction in zein, especially in the polypeptides of the 22kDa zein class. This reduction results from a lower rate of transcription of zein genes, particularly those of the 22kDa class. Available data have also suggested the importance of source supply in regulating zein synthesis in maize endosperm (Balconi, C et al., Plant Sci. 73:1-9, 1991). To study the effects of N nutrition upon endosperm development, normal and o2 maize endosperms of the W22 inbred line were grown in vitro for 5 days on a solid medium containing different N amounts (Table 1). A comparison between endosperms grown to maturity in vitro and under field conditions (in vivo) was also done.

Table 1. Culture media. All media contained 0.4mg/l thiamine, 100mg/l inositol, 30g/l sucrose, 8g/l agar and salts as described in Nitsch and Nitsch (media 1-2-3) or without ammonium nitrate (media 4-5-6).
Medium Ammonium nitrate (mg/l) Asparagine (g/l)
1 720 -
2 720 0.02
3 720 4
4 - -
5 - 0.02
6 - 4

Increased dry weight accumulation by cultured endosperms and increased asparagine concentrations in the medium were correlated. The same trend was observed for zein accumulation; in particular, the o2 endosperms cultured in vitro on the medium with the highest N content reached a zein level virtually identical to that of normal endosperms grown in vitro on the same medium, and in vivo at maturity (Fig. 1). Moreover, while o2 endosperms grown in vivo were much less efficient in accumulating the 22kDa fraction of zeins, when cultured in vitro they were capable of restoring a nor-mal synthesis of 22kDa zein fraction. Analyses of zein and b-32 mRNA levels in the wildtype and o2 endosperms also suggested that the expression of these RNAs is dependent upon the amounts of N supplied to the media. In conclusion, our data indicate that the zein synthesis system is subject to regulation by the levels of N provision, in particular asparagine, at the early stages of endosperm development. However, the short duration of endosperm culturing did not allow us to check if this feature is also maintained at later stages of development. Because zein gene expression and zein accumulation appeared to be regulated in response to amino acid supply at the level of gene transcription and/or stability of the mRNAs, this result raises the possibility that this type of metabolic control may be active at early stages of endosperm development, when N provision is not a limiting factor. One possibility is that the O2 gene, and consequently the expression of the 22kDa zeins, may be activated only later during the phase of rapid zein synthesis (from 15 to 30 DAP) and under conditions of amino acid starvation. In the yeast Saccharomyces cerevisiae, it is well documented that the expression of the transcription factor GCN4 increases under conditions of amino acid starvation (reviewed in Hinnebusch, Microbiol. Rev. 52:248-273, 1988). Alternatively, at early stages of endosperm development it can be postulated that a transcriptional activator dependent on N-availability promotes zein and b-32 gene expression. These questions are currently being addressed in our laboratory.

Figure 1. Zein content (ug/mg d.w.) of the W22 wildtype (m, r) and W22 mutant (m, n) endosperms grown in vivo and in vitro.

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