Sugars modulate an unusual mode of control of the cell-wall invertase gene (Incw1) through its 3' untranslated region in a cell suspension culture of maize.

We show here that a cell-wall invertase encoded by the Incw1 gene is regulated at both the transcriptional and posttranscriptional levels by sugars in a heterotrophic cell suspension culture of maize. The Incw1 gene encoded two transcripts: Incw1-S (small) and Incw1-L (large); the size variation was attributable to different lengths in the 3' untranslated region. Both metabolizable and nonmetabolizable sugars induced Incw1-L RNA apparently by default. However, only the metabolizable sugars, sucrose and D-glucose, were associated with the increased steady-state abundance of Incw1-S RNA, the concomitant increased levels of INCW1 protein and enzyme activity, and the downstream metabolic repression of the sucrose synthase gene, Sh1. Conversely, nonmetabolizable sugars, including the two glucose analogs 3-O-methylglucose and 2-deoxyglucose, induced greater steady-state levels of the Incw1-L RNA, but this increase did not lead to either an increase in the levels of the INCW1 protein/enzyme activity or the repression of the Sh1 gene. We conclude that sugar sensing and the induction of the Incw1 gene is independent of the hexokinase pathway. More importantly, our results also suggest that the 3' untranslated region of the Incw1 gene acts as a regulatory sensor of carbon starvation and may constitute a link between sink metabolism and cellular translation in plants.

Genetic evidence that the two isozymes of sucrose synthase present in developing maize endosperm are critical, one for cell wall integrity and the other for starch biosynthesis.

In maize, two paralogous genes, Sh1 and Sus1, encode two biochemically similar isozymes of sucrose synthase, SS1 and SS2, respectively. Previous studies have attributed the mild starch deficiency of the shrunken1 (sh1) endosperm to the loss of the SS1 isozyme in the mutant. Here we describe the first mutation in the sucrose synthase1 (Sus1) gene, sus1-1, and the isolation of a double recessive genotype, sh1 sus1-1. Combined data from diverse studies, including Northern and Western analyses, RT-PCR and genomic PCR, cloning and sequencing data for the 3' region, show that the mutant sus1-1 gene has a complex pattern of expression, albeit at much reduced levels as compared to the Sus1 gene. Endosperm sucrose synthase activity in sh1 sus1-1 was barely 0.5% of the total activity in the Sh1 Sus1 genotype. Significantly, comparative analyses of Sh1 Sus1, sh1 Sus1 and sh1 sus1-1 genotypes have, for the first time, allowed us to dissect the relative contributions of each isozyme to endosperm development. Starch contents in endosperm of the three related genotypes were 100, 78 and 53%, respectively. Anatomical analyses, which confirmed the previously described early cell degeneration phenotype unique to the sh1 Sus1 endosperm, revealed no detectable difference between the two sh1 genotypes. We conclude that the SS1 isozyme plays the dominant role in providing the substrate for cellulose biosynthesis, whereas the SS2 protein is needed mainly for generating precursors for starch biosynthesis.

The Miniature1 Seed Locus of Maize Encodes a Cell Wall Invertase Required for Normal Development of Endosperm and Maternal Cells in the Pedicel.

Collective evidence demonstrates that the Miniature1 (Mn1) seed locus in maize encodes an endosperm-specific isozyme of cell wall Invertase, CWI-2. The evidence includes (1) isolation and characterization of ethyl methanesulfonate-induced mn1 mutants with altered enzyme activity and (2) a near-linear relationship between gene/dose and invertase activity and the CWI-2 protein. In addition, molecular analyses showed that the cDNA clone incw2 maps to the Mn1 locus and differentiates the six ethyl methanesulfonate-induced mn1 mutants of independent origin into two classes when RNA gel blot analyses were used. We also report two unexpected observations that provide significant new insight into the physiological role of invertase and its regulation in a developing seed. First, a large proportion of total enzyme activity (~90%) was dispensable (i.e., nonlimiting). However, below the threshold level of ~6% of wild-type activity, the endosperm enzyme controlled both the sink strength of the developing endosperm as well as the developmental stability of maternal cells in the pedicel in a rate-limiting manner. Our data also suggest an unusually tight coordinate control between the cell wall-bound and the soluble forms of invertase, which are most likely encoded by two separate genes, presumably through metabolic controls mediated by the sugars.

Immunolocalization of a Unique Form of Maize Kernel Glutamine Synthetase Using a Monoclonal Antibody.

The pedicel (basal maternal tissue) of maize (Zea mays L.) kernels contains a physically and kinetically unique form of glutamine synthetase (GSp1) that is involved in the conversion of transport forms of nitrogen into glutamine for uptake by the developing endosperm (M.J. Muhitch [1989] Plant Physiol 91: 868-875). A monoclonal antibody has been raised against this kernel-specific GS that does not cross-react either with a second GS isozyme found in the pedicel or with the GS isozymes from the embryo, roots, or leaves. When used as a probe for tissue printing, the antibody labeled the pedicel tissue uniformly and also labeled some of the pericarp surrounding the lower endosperm. Silver-enhanced immunogold staining of whole-kernel paraffin sections revealed the presence of GSp1 in both the vascular tissue that terminates in the pedicel and the pedicel parenchyma cells, which are located between the vascular tissue and the basal endosperm transfer cells. Light staining of the subaleurone was also noted. The tissue-specific localization of GSp1 within the pedicel is consistent with its role in the metabolism of nitrogenous transport compounds as they are unloaded from the phloem.

Epistatic interaction and functional compensation between the two tissue- and cell-specific sucrose synthase genes in maize.

A tissue-specific epistatic mode of gene interaction was observed between molecularly homologous genes Sh1 and Sus1 (hereafter, Sh and Sus), encoding the sucrose synthase (SS) isozymes, SS1 and SS2, respectively. In Sh Sus genotype, both SS genes were expressed simultaneously and approximately equally in young seedlings; however, only the Sus-encoded SS2 protein was seen in the developing embryos. By contrast, the mutant sus genotype, lacking detectable levels of the SS2 protein in various tissues tested, showed expression of the Sh locus as judged by the detection of the SS1 protein in such embryos. Ectopic expression in embryos was seen from two separate Sh alleles, Sh-W22 and Sh'-5 (a revertant allele derived upon Ds excision from sh-m5933). In each case, the Sh expression at the protein level in embryos was unique to genotypes with the mutant sus gene. Based on the observed lack of phenotypic change in the sus mutant, we suggest that the ectopic expression of the Sh in otherwise Sus-specific tissues leads to functional compensation. There was no epistatic interaction of Sh and Sus at the RNA level as SS1 transcripts were detectable in both Sus and sus embryos. Thus, embryo specificity between the two SS genes was determined at posttranscriptional or at translational level of control. We surmise on the basis of these data that metabolic regulatory controls seem to override the normal constraints of tissue and cell specificity of the nonallelic isozyme genes to maintain efficient use of the pathways.

Tissue-Specific Expression and Anaerobically Induced Posttranscriptional Modulation of Sucrose Synthase Genes in Sorghum bicolor M.

We have used antibodies directed against the two sucrose synthase (SS) isozymes, and the cDNA clones corresponding to the two nonallelic genes in maize to describe sorghum (Sorghum bicolor) SS genes and their expressions at protein and RNA levels. Western blot analyses have shown evidence of two SS isozymes, SS1 and SS2, in sorghum; these were similar, but not identical, to maize isozymes in size, charge, subunit composition, and epitope specificities against both monoclonal and polyclonal antibodies. Tissue-specific distributions of isozymes and genomic Southern hybridization data are consistent with a hypothesis that the SS1 and SS2 isozymes are encoded by two nonallelic genes, designated here as Sus1 and Sus2, respectively. Northern blot hybridizations on root RNAs showed gene-specific transcript patterns and, as in maize, the SS2-specific transcripts were slightly larger than the SS1-specific transcripts. Interestingly, no difference in the size of the SS1 and SS2 polypeptides was detected. Anaerobic induction led to significant elevations in steady-state levels of both SS1 and SS2 transcripts, but there was no detectable increase in the levels of the SS proteins. Thus, both the SS genes in sorghum were significantly regulated at the posttranscriptional level; whereas in maize, only one of the two SS genes was affected in this fashion. Another difference between maize and sorghum SS isozymes was in endosperm-specific polymerization among the SS subunits. Unlike maize endosperm where only the two SS homotetramers are seen, sorghum endosperm showed five SS isozymes attributable to a random copolymerization of SS1 and SS2 subunits, presumably due to a simultaneous expression of both genes in the endosperm cells. Physiological and molecular bases of these differences between these two crop plant species remains to be elucidated.

Post-transcriptional control of sucrose synthase expression in anaerobic seedlings of maize.

We have examined post-transcriptional control of expression of the anaerobically induced sucrose synthase 1 (SS1) isozyme mRNA encoded by the shrunken (Sh) gene of maize (Zea mays L.). The SS1 transcript level is increased in maize seedling roots during anaerobiosis without a concomitant increase in the SS1 protein level. We show that the anaerobic SS1 RNA was loaded onto polyribosomes and that SS1 proteins produced by in vitro translation of polyribosomal RNA from anaerobic roots and immature kernels were indistinguishable based on abundance and apparent molecular weight. [(35)S]Methionine uptake in control and anaerobically stressed seedling roots indicated a detectable, but only slight, increase in radiolabel in the SS1 polypeptide as compared to the sucrose synthase 2 isozyme, SS2. However, this slight increase in [(35)S]methionine uptake did not contribute to a detectable increase in the steady state level of SS1 protein relative to SS2 protein. Chase experiments with unlabeled methionine indicated that SS1 protein was relatively stable in the anaerobic environment. From these results we conclude that SS1 protein was not rapidly turned over in the anaerobic environment and that expression of anaerobically induced SS1 transcripts was blocked at some step beyond polyribosomal loading.

Chromatin structure of integrated T-DNA in crown gall tumors.

We have investigated the chromatin structure of the integrated T-DNA in two N. tabacum crown gall tumor lines, and compared the results to those obtained in a previous study of the methylation patterns of these same integrated DNA sequences (Gelvin et al., Nucleic Acids Res. 11:159-174, 1983). The E9 octopine-type tumor contains a single copy of TL, whose transcription is essential for tumor maintenance, and 15-30 copies of TR, a non-essential region. The HT37#15 nopaline type teratoma contains a single copy of the nopaline T-DNA. All these integrated sequences can be found associated with nucleosomes, although the diffuse nature of the nucleosome bands on Southern transfers implies an 'open' chromatin conformation. In addition, all the sequences are more sensitive to digestion with deoxyribonuclease I than the bulk of the chromatin. We present evidence suggesting that, despite the previously published data that the majority of copies of the TR-DNA are highly methylated at the sequence CCGG whereas the TL-DNA is not, the majority of copies of the TR-DNA in the E9 tumor line are in the same chromatin conformation as TL. These data therefore suggest that most of the copies of TR-DNA are likely to be transcriptionally competent.

The nucleosome structure of the rRNA genes of some tumorous and nontumorous Nicotiana cell lines.

The nucleosome structure of the nuclear rRNA genes was investigated in a variety of tumorous and nontumorous Nicotiana tabacum cell lines, and in a genetic tumor produced by crossing Nicotiana langsdorffii with Nicotiana glauca. The rRNA genes from two unorganized octopine type crown gall tumors were found in an altered nucleosome conformation compared to those of the other cell lines and N. tabacum leaves. The altered nucleosome structure of the rRNA genes in the octopine type crown gall lines was not due to the tumorous state of the tissue, nor was it related directly to the morphology of the tumor. These two lines did have, however, a greatly reduced rRNA gene copy number. Several Eco R1 fragments homologous to the rRNA gene probe were preferentially lost from one of these tumor lines. The alteration of the nucleosome structure of the remaining rRNA genes in the octopine type crown gall tumors may result from rapid transcription necessitated by their reduced copy number.