BERGAMO, ITALY
Istituto Sperimentale per la Cerealicoltura Nitrogen-induced changes in the metabolism of in vitro grown developing kernels of defective endosperm maize mutant --Balconi, C, Bosio, D, Motto, M Although the biochemical mechanisms of both C and N synthetic processes in developing maize kernel have been generally elucidated (Moutot et al., Plant Physiol. 80:211-215, 1986; Pernollet et al., Plant Physiol. 80:216-222, 1986; Giroux et al., Plant Physiol. 106:713-722, 1994), the nature of the coordinated regulation of these still remains unclear. In order to improve the understanding of the mechanisms controlling starch and protein synthesis in maize endosperm, and to define genetic functions mediating the state of the metabolism on trascriptional regulation of endosperm-specific genes, the defective endosperm (de) mutant collection introgressed into the B37 inbred line (Manzocchi et al., Maydica 25:105-116, 1980) and the miniature (mn) (Lowe et al., Genetics 31:525-533, 1946) mutant, either grown in vivo or cultured in vitro on artificial media containing different levels of nitrogen nutrients, have been studied. For the in vivo evaluation, samples at 15 and 25 days after pollination (DAP), were collected. In order to evaluate a possible role of the cob and pedicel-placento-chalazal tissues (PPCh) in the amino acid supply, in the accumulation of zein and starch synthesis of developing caryopses, de and mn mutants were harvested at 10 DAP. Ears of the B37 wild-type were used as control. The ears were cut into blocks containing 10 kernels each as described by Gengenbach (Planta 134:91-93, 1977). The blocks were cultured on agar media plus salts as described by Nitsch and Nitsch (Science 163:85-87, 1969) containing NH4NO3 (0.72 g/l) as a source of nitrogen (N), 75 g/l sucrose and 0 (Medium A) or 4 (Medium B) g/l asparagine as a source of organic N. Kernels were incubated in the dark at 26 C until 15 or 25 DAP. Dry weights were determined for B37 and de mutants' endosperms, at different stages of development on the mother plant and after the in vitro culture. In vivo and in vitro endosperm samples were assayed for protein content (zeins, albumins plus globulins, glutelins), and for starch and soluble sugar content. In Table 1, zein content, as a percentage of dry matter (%/d.w.), of the B37 control, and of the de and mn mutant kernels grown in vivo or in vitro on the two media with different N content, at 15 and 25 DAP, are reported.

Table 1. Zein content as percent of dry matter (%/d.w) of in vivo and in vitro (medium A: 0 g/l asparagine and B: 4 g/l asparagine) grown kernels of the B37 + (wild-type), de mutant collection and mn mutant at 15 and 25 DAP.
 
  In vivo In vitro Medium A 0 g/l asparagine In vitro Medium B 4 g/l asparagine In vivo In vitro Medium A 0 g/l asparagine In vitro Medium B 4 g/l asparagine
Genotype 15 DAP 15 DAP (explant at 10 DAP + 5 days in vitro) 15 DAP (explant at 10 DAP + 5 days in vitro) 25 DAP 25 DAP (explant at 10 DAP + 15 days in vitro) 25 DAP (explant at 10 DAP + 15 days in vitro)
  Zeins (%/d.w) Zeins (%/d.w) Zeins (%/d.w) Zeins (%/d.w) Zeins (%/d.w) Zeins (%/d.w)
B37 + 2.70 2.89 4.22 4.20 2.15 4.25
de 1 3.04 2.60 2.10 4.53 2.30 3.52
de 6 2.56 2.20 3.40 6.61 4.41 4.65
de10 2.96 3.29 4.96 4.36 2.40 4.74
de 18 3.37 2.05 3.11 7.07 2.20 2.40
de21 4.96 1.90 2.10 9.34 2.30 2.71
de 37 3.07 1.95 2.78 4.92 6.23 7.23
de 110 2.08 3.74 3.18 6.96 3.20 3.69
de 116 3.01 2.56 5.97 7.90 2.17 3.20
de 241 1.99 2.25 1.91 8.16 2.06 2.57
de 246 1.30 2.43 3.19 5.53 2.07 4.71
mn 1 1.83 2.40 2.39 4.27 3.26 3.82

The data indicate that differences exist between B37 and its de version and among de mutants themselves. A comparison between the in vivo and in vitro developing endosperms, for a given genotype, suggested that the different de mutants respond in a peculiar manner to the different N levels. Differences for B37 and the mutants appeared evident relative to the utilization of organic and inorganic N for the protein synthesis. Asparagine in the in vitro culture media, induced, in almost all genotypes tested, an increase in zein content (as %/d.w.) at 25 DAP. More variability in zein accumulation, dependent on the nutrient supply in the media, was noticed at 15 DAP. At 25 DAP, de37 kernels grown in vitro on both media reached a zein content higher than that observed in field grown kernels at the same developmental stage. The B37 control, de10, de246, mn1, grown in vitro in the presence of asparagine, reached at 25 DAP a zein content similar or very close to that observed in vivo. All the other mutants showed on both culture media a zein content lower than that of field grown kernels. Studies are in progress to analyze if the in vitro culture method tested could mimic the development of defective maize endosperms grown in vivo; in addition these studies are aimed at understanding how the cob and PPCh tissues may be involved in determining the defective phenotype through a less efficient system of amino acid uptake, interconversion, and transport to the endosperm, in comparison with the control.
 
 

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