Tissue- and cell-specific expression of the two sucrose synthase isoenzymes in developing maize kernels

Summary The localization of the two known sucrose synthase isoenzymes of Zea mays L., sucrose synthase 1 and sucrose synthase 2, was studied during kernel development by indirect immunohistochemistry. These enzymes are encoded by the Sh and Sus genes, respectively. Since the antiserum used cross-reacts with both enzymes, tissue sections of Sh and sh kernels were compared. In the latter tissue no sucrose synthase 1 is expressed and thus the signal obtained was ascribed to sucrose synthase 2. We found that the isoenzymes are differentially expressed. While sucrose synthase 1 is expressed only in the endosperm, sucrose synthase 2 is found in almost all tissues of the kernel with cxpression levels specific for cell type and developmental stage. Sucrose synthase 2 is expressed strongly in the aleurone and subaleurone cell layers, where the signal detected is as strong as or even stronger than the sucrose synthase 1 signal in the inner endosperm. The distribution of the enzymes changes characteristically during development.     

Evidence that the two sucrose synthetase genes in maize are related

Summary Evidence is presented that the sucrose synthetase coding sequence at the Shrunken locus is distantly related to the sequence encoding a second, minor sucrose synthetase present in maize endosperm. Three doubly mutant sh bz strains lacking at least part of the Sh coding sequence produce an antigenically cross-reactive protein having the same electrophoretic mobility as the Sh-encoded, 92-kD sucrose synthetase monomer, but differing in primary structure. An mRNA is present in endosperm of mutants with deletions at the Sh locus that is weakly homologous to the Sh coding sequence and encodes a 92-kD protein precipitable with antiserum to sucrose synthetase. We conclude that the genes encoding the two different proteins are related.     

Evidence for plasma membrane-associated forms of sucrose synthase in maize

Plasma membrane fractions were isolated from maize (Zea mays L.) endosperms and etiolated kernels to investigate the possible membrane location of the sucrose synthase (SS) protein. Endosperms from seedlings at both 12 and 21 days after pollination (DAP), representing early and mid-developmental stages, were used, in addition to etiolated leaf and elongation zones from seedlings. Plasma membrane fractions were isolated from this material using differential centrifugation and aqueous two-phase partitioning. The plasma membrane-enriched fraction obtained was then analyzed for the presence of sucrose synthase using protein blots and activity measurements. Both isozymes SS1 and SS2, encoded by the lociSh1 andSus1, respectively, were detected in the plasma membrane-enriched fraction using polyclonal and monoclonal antisera to SS1 and SS2 isozymes. In addition, measurements of sucrose synthase activity in plasma membrane fractions of endosperm revealed high levels of specific activity. The sucrose synthase enzyme is tightly associated with the membrane, as shown by Triton X-100 treatment of the plasma membrane-enriched fraction. It is noteworthy that the gene products of bothSh1 andSus1 were detectable as both soluble and plasma membrane-associated forms.     

Transposable element Ds at the shrunken locus in Zea mays

Summary Maize DNAs isolated from wild type and from mutants caused by the insertion of transposable genetic element Ds at the gene encoding endosperm sucrose synthase (Sh) are compared in Southern blotting experiments by hybridization to Sh-cDNA cloned in pBR322. Differences observed between the DNAs of the wild type and the mutants indicate the presence of additional DNA at the Sh locus and/or DNA alterations that have occurred subsequent to the insertion of Ds. A double mutant exhibiting the recessive phenotype of both sh and the closely linked gene bz lacks DNA hybridizing to the probe and may be a deletion.     

The inheritance of rye seed peroxidases

Summary Genetic analyses were conducted on peroxidase of the embryo and endosperm of seeds of one open pollinated and six inbred lines of cultivated rye (Secale cereale L.), and one line of Secale vavilovii Grossh. The analyses of the individual parts of the S. cereale seed yield a total of 14 peroxidase isozymes. Isozymes m, a, b, c, d, e, f and g (in order from faster to slower migration) were found in the embryo plus scutellum, while isozymes 1, 2, 3, 4, 5 and 6 (also from faster to slower migration) were peculiar of the endosperm. S. vavilovii has isozymes m, c₁, d, e, f and g in its embryo plus scutellum, and isozyme 2 in the endosperm. Segregation data indicated that at least 13 different loci would be controlling the peroxidase of S. cereale. Isozymes a and b are controlled by alleles of the same locus, all the other loci have one active and dominant allele coding for one isozyme, and other null and recessive allele. The estimation of linkage relationships shows that five endosperm loci are linked, and tentative maps are shown. A possible dosage effect and the existence of controlling gene(s) for endosperm isozyme 4 is reported. All these data and the high frequency of null alleles found are discussed in relation to recent reports.     

DNase I hypersensitivity and expression of the Shrunken-1 gene of maize

The local chromatin structure of the Shrunken-1 (Sh) gene of maize was probed by analyzing DNase I hypersensitivity. Sh encodes the gene for sucrose synthetase, a major starch biosynthetic enzyme, which is maximally expressed in the endosperm during seed maturation. In addition to general DNase I sensitivity, specific DNase I hypersensitive sites were identified in endosperm chromatin that mapped near the 5 end of the Sh gene. The pattern of hypersensitive sites and their relative sensitivity were altered in other non-dormant tissues that produce little or no enzyme. However, some changes in chromatin structure appear to be independent of Sh gene expression and may reflect general alterations associated with plant development. The chromatin structure of several sh mutations, induced by Ds controlling element insertions, was also analyzed. Although the insertions perturbed expression of the gene, there were no notable effects on local chromatin structure.     

Intracellular immunolocalization of adenosine 5′-diphosphoglucose pyrophosphorylase in developing endosperm cells of maize (Zea mays L.)

We present immuno-electron microscopic evidence to show that ADPglucose pyrophosphorylase (AGP, EC 2.7.7.27) encoded by the Sh2 (shrunken 2) and the Bt2 (brittle 2) genes is localized to amyloplasts in developing endosperm of maize. The three AGP antibodies, including the two maize antisera, each raised against the Sh2encoded large or the Bt2-encoded small subunit and the spinach leaf protein, showed strong immuno-gold signals on developing starch grains in amyloplasts. The maize antibodies, but not the spinach, detected additional cross-reactivity sites to endosperm cell wall. Similar endosperm sections of the sh2-null mutant, lacking the Sh2-encoded subunit, yielded a drastic reduction in immuno signal on both starch grains and cell wall with the Sh2 anti-serum. However, the Bt2 and the spinach antisera showed no detectable difference between the sh2-null and the wild-type genotypes, except that the spinach antisera showed no reactivity to the cell wall in either of the two genotypes. Because the Bt2 epitope was readily detectable on the sh2-null starch grains, we suggest that the Bt2 subunit of this heteromeric enzyme is able to target itself to the organelle. The amyloplastic localization of the AGP protein in our studies is given additional significance by recent molecular data which indicate that full-length cDNA clones of the Sh2 and Bt2 genes show no cleavable transit-peptide signal sequence in their deduced aminoacid sequences. The observed disparity between the molecular and immunocytochemical data described here is discussed in the context of other proteins engaged in intracellular translocation with and without the known signal sequences.