Protease activity during germination in high and low protein inbreds and its reciprocal hybrids

--J. L. Magoja and A. A. Nivio

All those phenomena related to nitrogen accumulation in kernels are of great interest, whenever one tries to explain the different causes by which there are plants able to produce very different protein levels in their kernels. Although several investigations were carried out to clear up the subject, there are still numerous dark aspects linked to the genetic and physiological basis of nitrogen accumulation in kernels.

Our works--initiated in 1974--let us establish that the kernel protein content depends on the female parent genotype. As the plant has only got a unique genotype, all those grains belonging to the same ear have a similar protein content which relies on the capacity shown by the female parent to translocate amino acids up to the ear, during grain filling period. As we worked with two different inbreds the first with a high relative protein content NH) and the second showing a low relative protein content (BP), considering that protein content is a stable characteristic through a series of years (see Table 1), we could demonstrate that the F1 kernels derived from reciprocal crosses between them do not differ in their protein content from the inbred used as female parent.

These conclusions, according to the ones given by other authors, have already been published some years ago (Magoja, Rev. Fac. Agron. 44:203, 1978). Afterwards we could also verify that during the grain filling period, those lines with a low relative protein content have a lower free amino acid level in their leaves than the ones expressing a high relative kernel protein content.

Inbreds studied have a similar total nitrogen content in their vegetative organs, which let us deduce that the capacity of accumulating more protein in the kernel depends on a greater availability of free amino acids during grain filling period, which is related to a greater proteolytic activity in leaves and a higher translocation rate (Magoja and Streitenberger, XVI Reun. Nac. Fisol. Veg. La Plata, Arg, 1985).

It is probable that the proteolytic activity is strongly influenced by the endosperm protein content (available substratum). This fact would initially mask (during germination stage) the truly proteolytic activity, which is able to condition the hybrid genotype in other development stages of the plant (e.g., during grain filling period).

In connection with finding out whether the differential proteolytic activity among high and low protein lines can be detected in early stages (e.g., during seed germination) and then be able to predict the protein content in the next generation, an experiment using the BP, NH lines and their reciprocal F1 progenies was planned. Seeds of those lines and their hybrids were germinated in Petri dishes inside the oven at 28 C in the dark. Seven days after germination (for the lines) and five days after (for the hybrids), when all the coleoptiles came to the same length, the seedlings were dissected and the endosperm was taken away, from which the proteases were obtained.

The crude extract was incubated using casein as substratum, and the proteolytic activity was evaluated through quantification of the amino acids set free relative to non-incubated testers, expressed in micromols of glutamic acid per 100 milligrams of endosperm per hour. The results obtained at two different incubation times are given in Table 2.

The proteolytic activity depends on the incubation time although the detected differences among lines and hybrids keep relatively constant. The low relative protein content line (BP) and its hybrid resulting from crossing with the NH line (BPxNH) have lower proteolytic activity than the high relative protein content line (NH) and its hybrid resulting from the crossing with the BP line (NHxBP).

An average of seven ears of each participant (lines and hybrids) were analyzed, and a sample of grains taken at random was considered from each one. In spite of this, the results obtained show a considerable error which is greater at a minimum incubation time.

Although considering the error owing to the method, the results let us deduce that the proteolytic activity during germination is strongly associated with kernel protein content in the lines and reciprocal hybrids studied. Nevertheless this kind of evaluation could not be used to foretell the protein content of the grains harvested from those plants (the next generation), since the reciprocal hybrids might show a similar proteolytic activity when compared to their parents and in an intermediate level between them.

Table 1.  Whole kernel protein content (%) of BP, NH and its reciprocal hybrids.

Table 2.  Protease activity during germination (mM Glu/100mg.h-1) of BP, NH and its reciprocal hybrids.

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