MORPHOGENETIC RESPONSE, TRANSLOCATION, AND METABOLISM OF ROOT-APPLIED AUXIN IN PEA SEEDLINGS

PNL Volume 13 1981

RESEARCH REPORTS

Ingensiep, H. W., M. Merit, and II. L. Jacobsen

Institute of Genetics, University of Bonn, Federal Republic of Germany

Gene-dependent expression of phenotypic characters in plants seems to

be mediated by the various phytohormones. Since we have only a crude under-

standing of phytohormone action, model systems have been developed for evalua-

ting their activity in addition to their effects on morphology. For short-

term effects, cereal coleoptile served as a model tissue, while the study of

long-term biochemical effects was made possible mainly by using soybean

hypocotyl or pea epicotyl. At higher concentrations there seems to be no

culture system permitting comprehensive studies of the effects of different

auxins at the morphological, physiological, and biochemical level. In previous

studies we tried to investigate auxin action using in vitro systems (1), but

for a number of reasons we judged these systems to be unsuitable. So we

developed a system using intact pea seedlings and studied the different effects

of root-applied auxins (2,4-D, NAA, IAA). In our in vivo system we found

different morphogenetic responses detectable within one week, providing us

with sufficient amounts of tissue for investigation of auxin-protein inter-

action. It has been suggested that different modes of auxin action in pea

are connected with differences in translocation and metabolism of the res-

pective auxin (2). The present paper reports the effects of exogenously

applied auxins with respect to their uptake, transport, and metabolism in

intact pea seedlings. Concurrently, studies were carried out to detect changes

in protein level and to prove the existence of specific auxin-binding proteins

(3). Furthermore, the system may allow us to correlate possible different

auxin-sensitivities of pea mutants during germination with their genetic

background, thus obtaining some information, on the physiological level,

about the particular mutational event.

One-week-old etiolated pea seedlings grown on moist vermiculite were

carefully washed and placed on plastic grids in glass vessels. This arrange-

ment allows controlled uptake of the auxins by roots of intact seedlings (10

ml/seedling of 10~⁴M water solution of unlabeled auxin with 3 x 10⁵ dpm/ml

labeled 2,4-D and NAA, and about 1 x 10⁵ dpm/ml labeled IAA). After 24 h

-pulse of auxin in the dark, treated seedlings were placed on moist vermiculite

under a light/dark cycle of 16/8 h (2500 lux) for one week. Uptake of the

auxins was recorded by measuring loss of radioactivity (i.e. dpm or decompo-

sitions per minute) in the medium. The distribution of the auxins in the

seedling was determined after differential extraction from organs or segments

and by autoradiography of the dried seedlings with X-ray film. Ethanol

soluble extracts were analyzed by paper chromatography and on cellulose thin

layer aluminium sheets using different solvent systems.

Morphogenetic response: 2,4-D (10^-4M) inhibited natural differentiation

of pea seedlings treated with a 24 h -pulse by inducing abnormal growth in

root and shoot within one week (Figs. 1-4). NAA and IAA did not inhibit

natural differentiation of the seedlings under the same conditions (10^-4M)

and the seedlings are comparable to the untreated control although a slight

stimulation of normal root formation was observed. This raises the question:

What are the physiological reasons for these different morphogenetic responses?

PNL Volume 13 1981

RESEARCH REPORTS

The physiological results obtained in this system are summarized in

Table 1. In contrast to NAA and IAA, 2,4-D is characterized by a high mobility

and a low degradation in the intact plant. Uptake of 2,4-D by the roots was

about 6% after the 24 h- pulse, for NAA about 10%, and for IAA about 70% as

measured by loss of activity in the medium. In the case of 2,4-D, following

the pulse, radioactivity was accumulated in the root and during the following

culture period was transported into the shoot by the transpiration stream

as shown by differential extraction of segments and by autoradiographs. The

l_14c -activity of 2,4-D seems to be polar, having accumulated in the tip

regions of the lateral and adventitious roots, but being diffusely distributed

in stem and primary root tissue of the seedlings. There is no clear evidence

for degradation of 2,4-D. On the other hand NAA and IAA were characterized

by low mobility and high metabolic susceptibility of their molecules. After

one week most of the l-¹⁴C-activity from IAA or NAA still is fixed in the

primary root. Degradation or amino acid conjugation were detectable already

after the 24 h -pulse. The amount of free IAA is highly reduced by oxidation

and amino acid conjugation, while NAA seems to be mainly immobilized by con-

jugation. These results confirm earlier investigations (2, 4) and indicate

that the herbicidal effects of 2,4-D in pea depend on the endogeneously avail-

able amount of free unmetabolized auxin.

The data clearly demonstrate the advantages of the 2,4-D system used

in our investigations for studies of auxin-protein interactions in the higher

concentration range, 1) because of its pronounced morphogenetic response,

and 2) because of its less complex metabolic susceptibility.

Moreover, there are other methodological reasons favoring this culture

system: 1) The root-application of auxins has some advantages compared with

foliar-application often used. It allows easy control of uptake and calcula-

tion of accumulated auxin in the plants, and 2) last but not least this culture

system permits auxin action to be studied on the intact plant level, thereby

providing advantages over in vitro systems. Therefore, we think that the

2,4-D system is suitable for biochemical studies of auxin effects in the

herbicidal range. It was useful in investigating levels of response of cyto-

plasmatic and nuclear proteins and auxin-binding proteins. We assume that

auxin-mediating proteins could play an important role in long-term morpho-

genetic effects as demonstrated in the 2,4-D system.

1. Jacobsen, Fh et al. 1980. Tissue culture studies in Pisum sativum.

In: Plant Cell Cultures: Results and Perspectives. Elsevier, Amsterdam.

pp. 319-324.

2. Torrey, J. G. and R. Zobel. 1977. Root growth and morphogenesis. J_n:

The Physiology of the Garden Pea. Academic Press, London.

3. Jacobsen, H. J., et al. (in preparation).

4. Thimann, K. V. 1972. The natural plant hormones. I_n: Plant Physiology.

Vol. VIB. Academic Press, New York. pp. 3-365.



	RESEARCH REPORTS


	1.Effect of root-applied 2,4-D on pea seedlings :Inhibition of natu- ral differentiation within one week (untreated seedlings on the left). 2.Induction of abnormal adventitious roots and lateral roots after 2,4-D treatment(on the right).



	3.Induction of abnormal lateral root formation and callus-like structures after 2,4-D-treatment(left). 4.Effect of root-applied 2,4-D:radial expansion of stem tissue.