Asymmetric localization of seed storage protein RNAs to distinct subdomains of the endoplasmic reticulum in developing maize endosperm cells

Plant storage proteins are synthesized and stored in different compartments of the plant endomembrane system. Developing maize seeds synthesize and accumulate prolamin (zein) and 11S globulin (legumin-1) type proteins, which are sequestered in the endoplasmic reticulum (ER) lumen and storage vacuoles, respectively. Immunofluorescence studies showed that the lumenal chaperone BiP was not randomly distributed within the ER in developing maize endosperm but concentrated within the zein-containing protein bodies. Analysis of the spatial distribution of RNAs in maize endosperm sections by in situ RT-PCR showed that, contrary to the conclusions made in an earlier study [Kim et al. (2002) Plant Cell 14: 655-672], the zein and legumin-1 RNAs are not symmetrically distributed on the ER but, instead, targeted to specific ER subdomains. RNAs coding for 22 kDa alpha-zein, 15 kDa beta-zein, 27 kDa gamma-zein and 10 kDa delta-zein were localized to ER-bounded zein protein bodies, whereas 51 kDa legumin-1 RNAs were distributed on adjacent cisternal ER proximal to the zein protein bodies. These results indicate that the maize storage protein RNAs are targeted to specific ER subdomains in developing maize endosperm and that RNA localization may be a prevalent mechanism to sort proteins within plant cells.     

The maize regulatory locus Opaque-2 encodes a DNA-binding protein which activates the transcription of the b-32 gene

The maize locus, Opaque-2, controls the expression in developing endosperm of structural genes encoding a family of storage proteins, the 22 kd zeins, and an abundant albumin, termed b-32. It is shown that the promoter of the b-32 gene is activated in vivo in the presence of the O2 gene product and that the information necessary for this activation resides in a 440 bp DNA fragment containing five O2 binding sites (GATGAPyPuTGPu). Two of these sites are embedded in copies of the 'endosperm box', a motif thought to be involved in endosperm-specific expression, which is also represented in 22 kd zein promoters. The O2 protein is also shown to be capable of binding in vitro and activating in vivo, its own promoter.     

A new opaque variant of maize by a single dominant RNA-interference-inducing transgene

In maize, alpha-zeins, the main protein components of seed stores, are major determinants of nutritional imbalance when maize is used as the sole food source. Mutations like opaque-2 (o2) are used in breeding varieties with improved nutritional quality. However, o2 works in a recessive fashion by affecting the expression of a subset of 22-kD alpha-zeins, as well as additional endosperm gene functions. Thus, we sought a dominant mutation that could suppress the storage protein genes without interrupting O2 synthesis. We found that maize transformed with RNA interference (RNAi) constructs derived from a 22-kD zein gene could produce a dominant opaque phenotype. This phenotype segregates in a normal Mendelian fashion and eliminates 22-kD zeins without affecting the accumulation of other zein proteins. A system for regulated transgene expression generating antisense RNA also reduced the expression of 22-kD zein genes, but failed to give an opaque phenotype. Therefore, it appears that small interfering RNAs not only may play an important regulatory role during plant development, but also are effective genetic tools for dissecting the function of gene families. Since the dominant phenotype is also correlated with increased lysine content, the new mutant illustrates an approach for creating more nutritious crop plants.     

A wheat genomic DNA fragment reduces pollen transmission of maize transgenes by reducing pollen viability

A genomic DNA fragment from wheat carrying the Glu-1Dx5 gene has been shown to exhibit reduced pollen transmission in transgenic maize. To localize the region of the DNA fragment responsible for this reduced pollen transmission, we produced transgenic maize plants in which the wheat genomic DNA proximal to the 1Dx5 coding sequence was replaced with the maize 27 kDa gamma-zein promoter. Like the wheat promoter-driven Glu-1Dx5 transgene, this zein promoter-driven transgene functioned to produce 1Dx5 in maize endosperm. However, with the zein promoter-driven transgene, pollen transmission of the transgene loci was normal in most self- and cross-pollinations. We concluded that the wheat genomic DNA proximal to the wheat 1Dx5 coding sequence was required for reduced pollen transmission of the transgene in maize. In two of four transformation events of the wheat promoter-driven construct examined, pollen exhibited two morphological classes. In one class, pollen was normal in morphology and displayed average viability, and in the second, pollen was reduced in size and did not germinate on artificial media. DNA from the transgene was detectable in mature pollen from plants with reduced pollen transmission of transgene loci. To explain these observations, we hypothesize that elements within the transgene construct interfere with pollen development. We demonstrated that the wheat genomic DNA fragment can be used to control pollen transmission of an herbicide resistance transgene genetically linked to it. The wheat genomic DNA fragment may contain elements that are useful for controlling pollen transmission of transgene loci in commercial maize grain and seed production.