Some effects of minor nutrients on the growth and metabolism of plants

Investigations are described which were carried out to analyse the way in which certain mineral element deficiencies restrict the growth and development of plants. The plant system used in this work was excised pea roots grown in sterile culture media, and the deficiencies studied were those of iron, magnesium and molybdenum.
Growth was measured at the cell level and related to other characteristics of the system; two different experimental designs being employed to assess the effects of deficiencies. In the first, roots were grown in full nutrient and in deficient media and growth was measured on samples taken after growing periods of 0, 3, 5, 7, 9 and 11 days; while in the second, roots were grown for 7 days in full nutrient and in deficient media and serial one centimetre sections taken from these roots were compared. The first approach assessed the effects of the deficiency on overall growth, and the second gave an indication of the effects of the deficiency on the longitudinal differentiation of pea roots.
Both experimental approaches were employed when examining iron and magnesium deficient roots, but only the second when examining molybdenum deficient roots. Roots deficient in iron and magnesium were obtained by culturing tips cut from germinated seeds in deficient media, but two successive tip passages were necessary to obtain roots deficient in molybdenum.
Growth was assessed basically in terms of length, cell volume, protein nitrogen, and rate of oxygen uptake. However with iron deficient roots measurements of invertase activity, sensitivity of the oxygen uptake to cyanide, and the frequencies of cells in the different stages of division were also made. The techniques involved in the culture of deficient and full nutrient roots, and the analytical techniques are described.
It has been shown that iron deficiency markedly affects the growth and development of excised pea roots. Growth in terms of length and cell number per root is stopped after 7 days and no further increases occur between days 7 and 11. Although iron deficiency stops cell division, measurements made at day 7 indicate that this deficiency does not restrict the process of cell expansion. In fact 7 day old iron deficient roots carry larger cells in the terminal centimetre than full nutrient roots. By 11 days the iron deficient roots have a pronounced swelling at the terminal end, and it is suggested that this is brought about by an abnormal expansion of the cells in the lateral direction.
Some cells containing mitotic figures are present in the tips of 7 and 11 day old iron deficient roots. However there are fewer cells in the division stages of prophase and metaphase and practically no cells in the stages of telophase and anaphase in the deficient roots when comparisons were made with full nutrient roots.
The protein nitrogen content of iron deficient roots is lower than that of full nutrient roots at day 7, but there is a considerable increase in both deficient and full nutrient roots between days 7 and 11. The trend of the derived quantity, average protein nitrogen content per cell, is the same in both groups of roots up to day 7, but from day 7 to day 11 it increases sharply in the deficient roots but does not change in the full nutrient roots. This result indicates that cell division was not stopped in the deficient roots by a shortage of protein nitrogen as such.
At the day 7 stage the distribution of protein nitrogen along the length of deficient roots is different to that in full nutrient roots. The front sections of the deficient roots contain an increased content and the back sections a decreased content when compared with full nutrient roots. On a per cell basis the situation is the same, as the cells in the front sections of deficient roots have a higher average protein content and those in the back sections a lower content when compared with the cells of full nutrient roots.
The accumulation of protein nitrogen in the front sections of iron deficient roots is most probably associated with the cessation of active cell division in the meristem. Evidence is available which suggests that under normal conditions the formation and development of cells in the apex of the root is dependent on substrates synthesised in the mature regions of the root and translocated forward. It is considered that in iron deficient roots precursors of protein are no longer removed by the demands of the meristem and they condense to form protein in the regions adjacent to the apex.
Invertase activity per unit protein nitrogen is the same in both full nutrient and iron deficient roots at all stages. Further, there is no difference in invertase activity when the corresponding sections of full nutrient and deficient roots are compared at day 7. It is clear that in this one respect the protein of iron deficient roots is similar to that of full nutrient roots.
The rate of oxygen intake per root of iron deficient roots is lower than that of full nutrient roots at the early day 3 stage, but there are large increases in the rates in both deficient and full nutrient roots between days 3 and 11. It is of some significance that iron deficiency clearly reduces the rate of oxygen uptake at a stage before the process of cell division is stopped. On a per unit protein nitrogen basis the rate of oxygen uptake of deficient roots is lower than that of full nutrient roots after day 3. It is suggested that the effects of days 3 and 5 are a direct effect of iron deficiency but the effects at days 9 and 11 are influenced by the fact that cell division stops at day 7. The results from 7 day roots show that the effect of iron deficiency in reducing the rate of oxygen intake per unit protein nitrogen is confined to the front three sections of the root as iron deficiency does not alter the rates in the back three sections.
Iron recovery experiments show that iron deficient roots 7, 9 and 11 days old can resume cell division and grow when they are transferred to a full nutrient medium. It is of interest that in these experiments the recovery in terms of an increased rate of oxygen uptake is greater than the recovery in terms of length and protein nitrogen.
Experiments in which the rate of oxygen uptake of deficient and full nutrient roots were measured in the presence and absence of cyanide show that in both groups of roots there is a large fraction of the respiration insensitive to cyanide. The activity of this cyanide insensitive system increases considerably from day 3 to day 11 in both the full nutrient and iron deficient roots. Increases, after day 3 in the activity of this cyanide insensitive system, which would not contain iron, account for the large increase in the total rate of oxygen uptake of iron deficient roots between days 3 and 11.
The activity of the cyanide sensitive system involved in respiration decreases in both groups of roots between days 0 and 5. It increases from day 5 to 11 in full nutrient roots, but does not increase in deficient roots over this period. That synthesis of a cyanide sensitive system involved in respiration stops at or about the same stage as cell division in iron deficient roots is considered to be highly important.
This cyanide sensitive system most probably corresponds to the iron containing cytochrome/cytochrome oxidase system, and there is other circumstantial evidence that this system is important in the process of cell division. It is important to note that the activity of the cyanide sensitive system was the same in the tips of deficient and full nutrient roots at the day 7 stage. It may be that a certain minimum level of activity per cell is necessary to maintain division; a slight reduction stopping cell division completely, but not being capable of detection by the method of measurement. However, although there is a correlation between the activity of a cyanide sensitive system involved in respiration and the rate of cell division this correlation may not be causal, and indeed iron may be important in other reactions essential for cell division and growth.
The experiments involving magnesium deficiency have established that the deficiency restricts growth in terms of length after 7 days. There is no abrupt cessation; growth in length continues to occur in the deficient medium after 7 days although the differences between the control and the deficient series also continues to increase. This deficiency reduces the rate of cell division but it does not stop division over the experimental period of 11 days. Seven day magnesium deficient root tips contain approximately the same number of cells as full nutrient tips and even at 11 days the deficient roots have an apparently normal apical meristem. There were no differences between the average volumes of the cells in the corresponding sections of magnesium deficient and full nutrient roots at day 7 and it is clear that this deficiency does not affect the process of cell expansion.
The protein nitrogen content of deficient roots was lower than that of full nutrient roots at days 7, 9 and 11, but on each occasion the reduction in protein nitrogen was equal to the reduction in cell number, so that the average protein nitrogen content per cell was the same in the both groups of roots. The same situation was found at 7 days in successive sections taken along the length of deficient and full nutrient roots, and this deficiency clearly does not affect the average protein nitrogen content per cell. Magnesium deficiency produces an organised slowing down of growth and it is possible that here the rate of cell division may be controlled by the rate of protein synthesis.
The rate of oxygen uptake per root in deficient and full nutrient roots shows the same general trend as that for cell numbers and protein nitrogen, however on a per unit protein nitrogen basis the rate of oxygen uptake is significantly lower in the deficient roots from the early day 3 stage. There is only a slight increase in the rate of oxygen uptake per unit protein nitrogen from day 3 to day 11 in deficient roots, indicating that the protein of magnesium deficient roots less effectively catalyses the changes associated with oxygen uptake than that of full nutrient roots. The 7 day experiments show that this effect applies uniformly throughout the entire length of the roots as the rate of oxygen uptake per unit protein nitrogen is lower in all sections of the deficient roots when compared with the rates in the corresponding sections of full nutrient roots. The average rate of oxygen uptake per cell is reduced at practically all stages, and also along the entire length of the deficient roots.
It is known that magnesium is an activator of many enzyme systems involved in the metabolism of carbohydrates, and it is also an activator of enzymes concerned with the transfer of energy in the cell. It is probable that a deficiency of magnesium restricts one or a number of these reactions and this is the basis of the effect on rate of oxygen uptake and growth. The essential feature of magnesium deficiency is that it produces an organised slowing down of growth.
The experiments in which the effects of molybdenum deficiency were studied are only preliminary, but they do show that it is possible to obtain roots deficient in this element if at least two successive tip passages are made. Seven day molybdenum deficient roots are shorter, have a lower cell number, protein nitrogen content, and rate of oxygen uptake than full nutrient roots of the same age. The reduction in cell number is equal to the reduction in protein nitrogen and the average protein nitrogen content per cell is not affected by a deficiency of molybdenum. In view of the known importance of molybdenum in the metabolism of nitrogen it is suggested that the rate of cell division in molybdenum deficient roots may be controlled by the rate of protein formation. The overall rate of oxygen uptake per unit protein nitrogen was reduced in 7 day molybdenum deficient roots and this preliminary data suggests that the rate is reduced in practically all sections of the deficient roots. This deficiency in contrast to those of iron and magnesium brings about a reduction in the average volume per cell. The volume of the cells in all sections of the deficient roots are lower than those of the cells in the corresponding section of full nutrient roots.