Recombinants lacking in detectable levels of both sucrose synthases are functionally normal

The two sucrose synthase isozymes, SS1 and SS2, encoded by the Sh and Ss2 loci respectively, have been described previously (Chourey, MGG 184:372,1981; Chourey & Still, Proc. Intl. Genetics Symp. p. 207, ed. Reddy & Coe, Oxford & IBH Publ., 1983; Echt & Chourey, Plant Phys. 79:530, 1985). All spontaneous sh mutants analyzed so far and a sh deletion mutant (sh bz-m4) show only the SS2 protein; the SS1 protein is completely lacking in the mutants. Western blot analyses show that both the Sh and Ss2 genes are expressed in the wild type (Sh) genotype leading to either both homotetramers (S1S1S1S1 and S2S2S2S2) as in the developing endosperm cells, or a total of 5 isozymes (3 heterotetramers, S1S1S1S2, S1S1S2S2, and S1S2S2S2, and 2 homotetramers) as in the seedling cells (Chourey et al., MGG 203:251, 1986). The Sh-r5 revertant described previously (Courage-Tebbe et al., Cell 34:383, 1983) has led to isolation of the first Ss2-null allele (Chourey et al., 1986 CSH meeting, p.65 & manuscript submitted), a single protein band of SS1 electrophoretic mobility is seen in all tissues of this revertant. Because the Ss2-null (i.e. ss2 ss2) expression did not segregate independently from the homozygous Sh-r5 phenotype, we suggested that both the SS encoding genes are on chromosome 9. Two independent molecular mapping studies have now mapped Ss2 to 9L (McCarty et al., PNAS, 83:9099, 1986; Gupta et al., Plant Molec. Biol., 1987, in press).

The demonstrated loose linkage between Sh and Ss2 genes (approx. 30 to 40 map units) led to the obvious question: can the double homozygotes lacking both SS1 and SS2 proteins be isolated? The data given below indicate that such recombinants can be isolated and surprisingly are functionally normal. Because the Ss2-null expression is not associated with any detectable plant/seed phenotype, individual sh segregants from the cross given below were examined by Southern and Western analysis. The usefulness of genomic Southern tests in such analysis is related to the fact that the Ss2-null allele in Sh-r5 strain is unique (manuscript submitted). Briefly, the EcoRI genomic digests of the ss2/ss2 (null) homozygotes yield 2 fragments (~23kb and 8kb), the Ss2/Ss2 homozygous allele in sh bz-m4 stock shows a ~ 16kb fragment and the Ss2/ss2 heterozygote shows a combination of the 3 fragments.

We made the following cross: Sh-r5/Sh-r5,ss2/ss2 X sh/sh,(bz-m4),Ss2/Ss2. The sh segregants on the F2 ears were selected, genomic DNA from a certain number of such individual sh seedlings was EcoRI restricted and examined for Ss2 hybridizing fragments.

Number of individual F2 sh seedlings
Ear # 16 kb 16 + 23 + 8 kb 23 + 8 kb Total
1 13 17 5 35
2 10 14 4 28
3 9 12 1 22
Total 3 32 43 10 85

Root extracts from some of the individual seedlings used for the above Southern tests were also examined by Western blot analysis for the SS protein. A total of 4 seedlings showing the 23 + 8kb fragments (the presumptive ss2 ss2 genotype) from two separate ears showed no detectable levels of SS protein. This entirely unexpected observation leads us to suggest that the lack of detectable levels of SS1 and SS2 proteins is not associated with impaired functions relating to development, maturation or germination of the kernel. Similar immunoblot tests on root extracts from several individual seedlings of the other 2 classes (the presumptive Ss21Ss2 and Ss2lss2) showed, as expected, a single SS2 protein band. Enzymatic analyses on these genotypes have not been done because routine tests have shown that Western blots are far more sensitive than the assay for enzyme activity, particularly in seedling extracts where the enzyme activity is highly labile.

In a second set of experiments, a F2 sh/sh plant from a sibbed progeny of the ear #1 was identified as having the 23 + 8kb Ss2 hybridizing fragments (i.e. putative ss2/ss2, it was not typed for SS protein expression). It was used as a male parent in the following cross (lack of flowering synchrony prevented selfing): Sh/Sh-r5,Ss2/ss2 X sh/sh, putative ss2/ss2 (The female parent was also heterozygous for Gl15/gl15 but is disregarded here). Four possible genotypes of the seedlings derived from this cross and the predicted SS protein pattern on Western blots are:
  SS bands
Sh/sh,Ss2/ss2 5
Sh/sh,ss2/ss2 1
Sh-r5/sh,Ss2/ss2 5
Sh-r5/sh,ss2/ss2 1

A total of 30 F1 individual seedlings have been typed so far by Western analysis. A segregation of 17 seedlings for 5 bands and 13 for 1 band pattern has led us to conclude that the male parent in the above cross was indeed of the ss2/ss2 genotype. The important conclusion from this analysis is that plants lacking in detectable levels of both the sucrose synthases are not only nonlethal but are functional in completing the life cycle. Efforts are in progress to obtain a SS-null (sh/sh, ss2/ss2) stock so as to better understand the physiological role of sucrose synthase in the maize plant.

Prem S. Chourey

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