--Jonathan D. Tom and Julia Bailey-Serres*

*Present address: UC Riverside

Two PGD isozyme subunits, PGD1 and PGD2, are regulated in tissue specific fashion. We used PGD nulls to examine the localized distribution of cytosolic 6-PGD isozymes in the mature scutellum. A PGD1+PGD2 double null was constructed by crossing plants which were homozygous for Pgd2-null but wild-type at Pgd1 to plants homozygous for Pgd1-null but wild-type at Pgd2. In the F2 progeny, we observed the expected 9:3:3:1 segregation of the four different 6-PGD isozyme dimer phenotypes: wild-type, PGD2null, PGD1null, PGD1+PGD2 double null. Fresh cross-sections of mature scutella from the four phenotypes were stained in situ for 6-PGD activity. The 6-PGD stained scutella were compared to scutella of the same genotype which had been stained without 6-phosphogluconate substrate (Fig. 1 Control column) and for alcohol dehydrogenase (ADH) (Fig 1, ADH column). In scutella from the inbred B73 (Pgd1-3.8, Pgd2-5) and Pgd2-null, we observed specific PGD1 activity associated with pro-vascular bundles (See arrows Fig. 1A and 1B). In contrast, the PGD2 activity observed in scutella from Pgd1-null was uniform throughout the interior of the scutellum (Fig. 1D). In scutella from PGD1+PGD2 double nulls, no detectable PGD activity was observed (Fig. 1C). Since there are PGD1.PGD2 heterodimers in scutellar extracts (data not shown), it is likely that PGD1 is expressed in non-vascular as well as vascular tissue. Therefore, in mature scutella, PGD1 expression appears to be most intensely localized in the pro-vascular bundles whereas PGD2 occurs only in the non-vascular tissue.

The 6-PGD isozyme dimer patterns from primary roots, secondary roots, mature scutella, embryo axis, pollen and leaves were analyzed by native-polyacrylamide gel electrophoresis. Based on isozyme dimer ratios, we observed a variation in the tissue and organ-specific distribution of cytosolic 6-PGD isozymes among the tissues analyzed in the inbred B73 (data not shown). Using PGD nulls, we also demonstrated that the relative ratios of PGD1 and PGD2 isozymes were proportional to gene copy number (data not shown). In a moderate environment, the PGD1+PGD2 double null exhibited no obvious reduction in vigor, seed set, time to maturity, pollen viability, or germination when compared to its siblings. Even under anaerobic stress, we observed no reduction in germination for the PGD1+PGD2 double null as compared to PGD-positive seedlings. However, we are uncertain whether the pentose pathway was stressed under anaerobiosis. Since the PGD1+PGD2 double null's growth and development resembles that of wild-type, cytosolic 6-PGD activity appears to be dispensable. 6-PGD activity is localized the plastid as well as in the cytosol (Gottlieb, Science, 216: 373-380, 1982). Plastid isozymes may provide sufficient 6-PGD activity for the survival of plants that have no cytosolic PGD activity.

Work performed in the Freeling lab.

Figure 1. Fresh cross-sections of mature scutella were stained in situ for PGD, ADH, or PGD without 6-phosphogluconate substrate (control) Since no activity associated with the pro-vascular bundles in mature scutella was observed for ADH or PGD control activity stains, PGD activity exhibits tissue specificity. The arrows indicate the mature vascular bundles in scutella. (A) Scutella from the inbred, B73 (Pgd1-3.8:Pgd2-5) were understained to show mature vasculature. (B) Scutella from a recessive Pgd2-null mutant (Pgd1-3.8: Pgd2-null). Note the PGD1 activity localized in the vascular bundles (see arrows) in (A) and (B). (C) Scutella from a recessive Pgd1-null/Pgd2-null double null mutant (Pgd1-null: Pgd2-null). No activity was detected. (D) Scutella from a recessive Pgd1-null mutant (Pgd1-null: Pgd2-5). Note the absence of PGD1 activity associated with vascular bundles. Scutella in (B), (C), and (D) are progeny derived from the same parents.

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