Detailed analysis of the aerobic and anaerobic tissue distribution of ADH1 activity in the primary root

--Julie Vogel, John Fowler and Michael Freeling

It has long been known that alcohol dehydrogenase-1 (ADH1) is expressed in the aerobic primary root (Schwartz, MGG 127:215, 1971), and that the level of enzyme activity in the root as a whole is induced at least 10-fold upon anaerobiosis (Freeling, Genetics 67:411, 1973). More recently, Christie Williams examined in more detail the distribution of ADH among a number of tissues of the seedling primary root as part of her Ph.D. thesis work in this laboratory ("Organ-specific Expression of ADH in Drosophila melanogaster and Zea mays", UC-Berkeley, 1987). Using in situ staining of longitudinal root sections to assay ADH enzyme activity, Williams found that, under aerobic conditions, ADH1 is expressed only in the root cap and the stele (probably only the inner parenchymous tissue). However, upon 18hr of anaerobic treatment, not only was the staining more intense in these same tissues, but de novo ADH1 activity was apparent in the cortex, meristem, epidermis, and some longitudinal elements of the vascular system. We have extended this initial analysis, using both longitudinal and transverse sections, and we have made a number of further observations about the tissue distribution of ADH1 in the primary root. All of our results are controlled in two ways. First, we compare staining patterns with and without ethanol to make sure that our NADH source is indeed from the ethanol-driven ADH reaction. Second, since we have null alleles for both Adh1 (which encodes ADH1 subunits) and Adh2 (which encodes a very low level of ADH activity that is below the level of detection of this assay), we know that ADH1 subunit activity alone is being monitored.

First, we observed that the same anaerobic pattern of ADH1 expression could be seen after only 8hr of anaerobic treatment, performed by immersing 5-day-old seedlings in water saturated with bubbling argon gas. However, at this 8hr time point, the two different maize Adh1 genotypes examined, 1S (of the 1s2p inbred line) and 1FB73 (of the B73 inbred line), exhibited consistent differences in the apparent levels of ADH1 induction in the various root tissues. Although the absolute levels of ADH activity could not be quantified by this visual assay, the level of staining in B73 appeared to be at least 2-fold greater in all induced tissues than in 1s2p. It is possible that cis-acting sequences at these Adh1 alleles are responsible for these quantitative differences, or, alternatively, that one or more unlinked "modifier" genes differing in these lines may confer such phenotypic variation. These two possibilities are to be tested.

In addition, our tissue distribution analysis has revealed that ADH1 is anaerobically induced, apparently de novo, in the endodermis, the innermost cell layer of the cortex that directly surrounds the pericycle. However, activity in this cell layer is most pronounced only in the elongation region of the root; the endodermis does not stain appreciably in the meristematic region nor in the root maturation zone. We also observed ADH1 induction in the epidermis, but only within a limited region of the root, which appears to correspond roughly to the elongation zone. In our 5-day-old anaerobic seedlings, with roots 3-5cm long, epidermal ADH1 activity was detected only within a distance of ~0.5-0.7cm from the root tip. There were no root hairs in the elongation zone, and hairs in the older portion of the root, like the rest of the surrounding epidermal cells in this region, did not stain for ADH.

The root cap and stele express ADH1 activity under normal growth conditions; all other tissues or tissue regions induce ADH1 in response to anaerobiosis. We do not know the functional significance (evolutionary advantages) of these observations.

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