A point mutation at the Miniature1 seed locus reduces levels of the encoded protein, but not its mRNA, in maize.

We report here on the molecular nature of an EMS-induced mutant, mn1-89, a leaky semidominant allele of the Miniature1 (Mn1) seed locus that encodes a seed-specific cell wall invertase, INCW2. The mn1-89 locus specifies normal levels of the Incw2 transcript but extremely low levels (about 6% of normal) of the protein and enzyme activity are expressed. Sequence analysis of Incw2 clones derived from the parental Mn1 and the mutant genotypes shows a C to T transition in the mn1-89 allele, leading to a single amino acid alteration (proline to leucine) near the C-terminus of the mutant INCW2 protein. Although this change is not in the catalytic domain, putative N-glycosylation sites, or the beta-fructosidase motif, it does lie in a motif that is well conserved among all plant invertases and related fructosyltransferases. On the basis of these genetic in planta data, we believe we have identified a proline residue in a hitherto unknown GPFG motif as critical for the stability of such proteins. The single base change (C to T) also leads to the elimination of a BglI restriction site in the mutant allele. Indeed, BglI restriction digests of genomic DNAs from mn1-89 and Mn1 genotypes show one and two fragments, respectively. Sequence analysis of RT-PCR-derived endosperm Incw clones from mn1-1 (the reference allele) seeds predict five amino acid substitutions relative to Mn1. Whether or not these sequences are encoded by the mn1-1 locus or another non-allelic Incw gene in the maize genome remains to be elucidated.

Characterization of two members of the maize gene family, Incw3 and Incw4, encoding cell-wall invertases.

Two maize putative cell-wall invertase genes (Incw3 and Incw4) have been isolated by screening a genomic DNA library (Zea mays L. W22) using the cDNA probes encoding the two maize cell-wall invertases Incw1 and Incw2. The Incw3 and Incw4 genes contain six exons/five introns and five exons/four introns, respectively. The protein sequences deduced from both genes revealed a beta-fructosidase motif and a cysteine catalytic site known to be conserved in invertase genes. A detailed analysis of the protein and nucleotide sequences provides evidence that the Incw3 and the Incw4 genes encode putative cell-wall invertases. Furthermore, the isoelectric point deduced from the INCW4 protein sequence suggested that the Incw4 gene may encode a unique type of cell-wall invertase unbound in the apoplast. Gene expression studies using RT-PCR and in-situ RT-PCR hybridization showed that the Incw3 expression is organ/tissue-specific and developmentally regulated. In contrast, the Incw4 gene is constitutively expressed in all vegetative and reproductive tissues tested.

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 maize glutamine synthetase GS1-2 gene is preferentially expressed in kernel pedicels and is developmentally-regulated.

The pedicel region of Zea mays kernels contains a unique form of maize glutamine synthetase (GS), GSp1. RNA blot analysis using GS gene-specific probes revealed that the expression of the GS1-2 gene was specific to the pedicel and that it increased in the kernels during development. This pattern of the maize GS1-2 gene expression is consistent with the tissue specificity of the GSp1 protein and suggests that it encodes the GSp1 isoform of maize GS.

Cloning of a novel maize endosperm-specific protein with partial sequence homology to a pollen surface protein.

A gene encoding a novel maize endosperm protein has been cloned and sequenced. The gene encodes a 43 135 Da polypeptide which is 42% identical over two segments of an alfalfa pollen protein sequence. The gene is expressed in developing endosperm tissue, and not in other tissues such as shoot, pollen, or embryo.

The Differential Expression of Sucrose Synthase in Relation to Diverse Patterns of Carbon Partitioning in Developing Cotton Seed.

Developing cotton (Gossypium hirsutum L.) seed exhibits complex patterns of carbon allocation in which incoming sucrose (Suc) is partitioned to three major sinks: the fibers, seed coat, and cotyledons, which synthesize cellulose, starch, and storage proteins or oils, respectively. In this study we investigated the role of Suc synthase (SuSy) in the mobilization of Suc into such sinks. Assessments of SuSy gene expression at various levels led to the surprising conclusion that, in contrast to that found for other plants, SuSy does not appear to play a role in starch synthesis in the cotton seed. However, our demonstration of functional symplastic connections between the phloem-unloading area and the fiber cells, as well as the SuSy expression pattern in fibers, indicates a major role of SuSy in partitioning carbon to fiber cellulose synthesis. SuSy expression is also high in transfer cells of the seed coat facing the cotyledons. Such high levels of SuSy could contribute to the synthesis of the thickened cell walls and to the energy generation for Suc efflux to the seed apoplast. The expression of SuSy in cotyledons also suggests a role in protein and lipid synthesis. In summary, the developing cotton seed provides an excellent example of the diverse roles played by SuSy in carbon metabolism.

Phosphorylation of serine-15 of maize leaf sucrose synthase. Occurrence in vivo and possible regulatory significance.

Experiments were conducted to determine whether sucrose synthase (SuSy) was phosphorylated in the elongation zone of maize (Zea mays L.) leaves. The approximately 90-kD subunit of SuSy was 32P-labeled on seryl residue(s) when excised shoots were fed [32P]orthophosphate. Both isoforms of SuSy (the SS1 and SS2 proteins) were phosphorylated in vivo, and tryptic peptide-mapping analysis suggested a single, similar phosphorylation site in both proteins. A combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and automated Edman sequencing analysis unequivocally identified the phosphorylation site in the maize SS2 protein as serine-15. This site was phosphorylated in vitro by endogenous protein kinase(s) in a strictly Ca(2+)-dependent manner. A synthetic peptide, based on the phosphorylation site sequence, was used to identify and partially purify an endogenous Ca(2+)-dependent protein kinase(s) from the maize leaf elongation zone and expanding spinach leaves. Phosphorylation of SuSy in vitro selectively activates the cleavage reaction by increasing the apparent affinity of the enzyme for sucrose and UDP, suggesting that phosphorylation may be of regulatory significance. Conservation of the phosphorylation site, and the sequences surrounding it, among plant species suggests that phosphorylation of SuSy may be widespread, if not universal, in plants.

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 loci Sh1 and Sus1, 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 both Sh1 and Sus1 were detectable as both soluble and plasma membrane-associated forms.

Sucrose Phosphate Synthase Expression at the Cell and Tissue Level Is Coordinated with Sucrose Sink-to-Source Transitions in Maize Leaf.

Immunohistological analyses for sucrose phosphate synthase (SPS) show that the protein is localized in both bundle-sheath cells (BS) and mesophyll cells (M) in maize (Zea mays) leaves. In young leaves, SPS protein was predominantly in the BS, whereas mature leaves showed nearly equal levels of signal in both BS and M. A cell-type-specific response was also seen in light and dark treatments. Dark treatments led to reduced signal in M; however, little or no change was detected in BS. We suggest that SPS in BS is engaged in sucrose biosynthesis by both photoassimilatory and starch turnover reactions in maize leaves. In addition, we suggest that the enzyme in BS may play a major role in the early biosynthesis of sucrose in young leaves. These cell-specific changes in expression in situ were in agreement with the estimates of extractable enzyme activity from isolated BS and M of mature leaves (R. Ohsugi, S.C. Huber [1987] Plant Physiol 84: 1096-1101). In contrast, western blot analyses did not show any significant changes in the levels of SPS protein in either young or mature leaves subsequent to similar dark treatments. It is interesting that the northern blot analyses indicate that the steady-state levels of SPS transcripts were markedly reduced after dark treatments of > 12 h. Overall, our results indicate that Sps gene expression in maize leaf is modulated at multiple levels of controls by both developmental and environmental factors.

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.

The Maize Invertase-Deficient miniature-1 Seed Mutation Is Associated with Aberrant Pedicel and Endosperm Development.

Genetic evidence is presented to show that the developmental stability of maternal cells in the pedicel at the base of maize seeds is determined by the genotype of the developing endosperm. An early degeneration and withdrawal of maternal cells from the endosperm of homozygous miniature (mn mn) seed mutants were arrested if mn plants were pollinated by the wild-type Mn pollen. Similarly, the stability of the wild-type, Mn mn, maternal cells was also dependent on whether or not these cells were associated with the normal (Mn) or the mutant (mn) endosperm on the same ear. Biochemical and cellular analyses indicated that developing mn kernels have extremely low (<0.5% of the wild type) to undetectable levels of both soluble and wall-bound invertase activities. Extracts from endosperm with a single copy of the Mn gene showed a significant increase in both forms of invertases, and we suggest it is the causal basis of the wild-type seed phenotype. Collectively, these data provide evidence that invertase-mediated maintenance of a physiological gradient of photosynthate between pedicel and endosperm constitutes the rate-limiting step in structural stability of maternal cells as well as normal development of endosperm and seed.

Mitochondrial genome variability in Sorghum cell culture protoclones.

Sorghum bicolor cv NK300 seedlings, a cell suspension culture, and five protoclone suspension cultures were compared for the occurrence of somaclonal variation by analysis of their mitochondrial DNA (mtDNA). Restriction digests of the mtDNA showed qualitative and quantitative variation of restriction fragments. Southern analyses were performed using a 14.7-kb EcoRI mitochondrial genome fragment and regions carrying mitochondrial protein coding genes, atpA, atp6, cob, and coxI as probes. These analyses revealed part of the 14.7-kb EcoRI region to be present as a repeat in planta, and to be hypervariable when cells were subjected to protoplast culture. All protoclones differed from each other, from the parental cell suspension culture, and from the seedlings in their mitochondrial genome arrangement. Seedlings of five independent sorghum accessions, unrelated to cv NK300, of diverse geographic origin showed conservation of this mitochondrial fragment. Southern analyses of the mtDNA showed no variation for genomic organization of the region carrying coxI, and atpA was identical in all the tissue culture lines. The atp6 gene was present as two copies in the seedlings, and one copy was rearranged upon tissue culture. The region carrying the cob gene was also found to be variant between tissue culture and seedling mtDNA. A substoichiometric 3.3-kb EcoRI cob fragment present in seedlings was amplified in the tissue culture lines. Protoclone S63 differed from the original suspension culture and remaining protoclones in that it had lost the 3.0-kb EcoRI band, the most abundant fragment in seedlings. A new set of fragments was detected in this protoclone. Northern analysis for the cob gene demonstrated altered transcript size in protoclone S63.

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.

Spatial and temporal expression of the two sucrose synthase genes in maize: immunohistological evidence.

We describe the spatial and temporal immunohistological distributions of the two sucrose synthases, SS1 and SS2, encoded by the Sh and Sus genes, respectively, in different parts of the maize plant. The two similar isozymes were differentially localized in developing endosperm cells through the combined uses of a shrunken (sh) mutant lacking the SS1 protein and the SS1 and SS2 antisera. The accumulation of SS1 protein always coincided with starch deposition in the Sh endosperm cells, whereas in the sh endosperm, the centrally located cells were lost at or during the most critical phase of starch biosynthesis. The SS2 specific cells, including aleurone layer and the basal endosperm transfer cells in both genotypes, were not associated with detectable starch deposition. Such heterogeneity was indicative of two cell types separable by gene expression, and of differential in vivo roles of the two isozymes in the endosperm. In young roots, the expression of both SS encoding genes was predominantly in the vascular cylinder region. These data fulfill a previous prediction, based on the genetic analyses, that the expression of the SS genes is spatially and/or temporally separated in endosperm cells but not in root cells.