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Summary Sorghum prolamins, termed kafirins, are categorized into subgroups a, b, and c. The kafirins are co-translationally translocated to the endoplasmic reticulum (ER) where they are assembled into discrete protein bodies that tend to be poorly digestible with low functionality in food and feed applications. As a means to address the issues surrounding functionality and digestibility in sorghum, we employed a biotechnology approach that is designed to alter protein body structure, with the concomitant synthesis of a co-protein in the endosperm fraction of the grain. Wherein perturbation of protein body architecture may provide a route to impact digestibility by reducing disulphide bonds about the periphery of the body, while synthesis of a co-protein, with known functionality attributes, theoretically could impact structure of the protein body through direct association and ⁄or augment end-use applications of sorghum flour by stabilizing s-sheet formation of the kafirins in sorghum dough preparations. This in turn may improve viscoelasticity of sorghum dough. To this end, we report here on the molecular and phenotypic characterizations of transgenic sorghum events that are downregulated in c- and the 29-kDa a-kafirins and the expression of a wheat Dy10 ⁄Dx 5 hybrid high-molecular weight glutenin protein. The results demonstrate that down-regulation of c-kafirin alone does not alter protein body formation or impacts protein digestibility of cooked flour samples. However, reduction in accumulation of a predicted 29-kDa a-kafirin alters the morphology of protein body and enhances protein digestibility in both raw and cooked samples.

Plastid number and morphology vary dramatically between cell types and at different developmental stages. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized in mesophyll and bundle sheath cells, which differentiate acropetally from the proplastid form in the leaf base. To develop visible markers for maize plastids, we have created a series of stable transgenics expressing fluorescent proteins fused to either the maize ubiquitin promoter, the mesophyll-specific phosphoenolpyruvate carboxylase (PepC) promoter, or the bundle sheath-specific Rubisco small subunit 1 (RbcS) promoter. Multiple independent events were examined and revealed that maize codon-optimized versions of YFP and GFP were particularly well expressed, and that expression was stably inherited. Plants carrying PepC promoter constructs exhibit YFP expression in mesophyll plastids and the RbcS promoter mediated expression in bundle sheath plastids. The PepC and RbcS promoter fusions also proved useful for identifying plastids in organs such as epidermis, silks, roots and trichomes. These tools will inform future plastid-related studies of wild-type and mutant maize plants and provide material from which different plastid types may be isolated.

Phosphoenolpyruvate carboxylase (PEPC) is a widely distributed metabolic enzyme among plant and prokaryotic species. In vascular plants, the typical PEPC is regulated post-translationally by a complex interplay between opposing metabolite effectors and reversible protein phosphorylation. This phosphorylation event is controlled primarily by the up-/down-regulation of PEPC-kinase (PpcK), an approximately 31-kDa Ser/Thr-kinase. As a sequel to earlier investigations related to PEPC phosphorylation in N(2)-fixing nodules of Glycine max, we now present a detailed molecular analysis of the PpcK multigene family in nodulated soybeans. Although the GmPpcK1-4 transcripts are all expressed throughout nodule development, only the nearly identical GmPpcK2/3 homologs are nodule-enhanced and up-/down-regulated in vivo by photosynthate supply from the shoots. In contrast, GmPpcK1 is a 'housekeeping' gene, and GmPpcK4 is a highly divergent member, distantly removed from the legume PpcK subfamily. Real-time qRT-PCR analysis indicates that GmPpcK2/3 are overwhelmingly the dominant PpcKs expressed and up-/down-regulated throughout nodule development, mirroring the expression properties of nodule-enhanced PEPC (GmPpc7). In situ RT-PCR investigation of the spatial localization of the GmPpcK1-4 and GmPpc7 transcripts in mature nodules is entirely consistent with this view. Complementary histochemical and related RNA gel-blot findings with nodulated, GmPpcK1/3 promoter::GUS-expressing T(2) plants provide direct experimental evidence that (i) PpcK gene expression is controlled primarily at the transcriptional level; and (ii) the contrasting expression properties of GmPpcK1/3 are conferred largely by regulatory element(s) within the approximately 1.4-kb 5'-upstream region. As a result of our multifaceted analyses of GmPpcK1-4, GmPpc7 and PEPC-phosphorylation in the soybean nodule, it is proposed that the GmPpcK2/3 homologs and GmPpc7 together comprise the key molecular 'downstream players' in this regulatory phosphorylation system within the mature nodule's central zone.

Through a single desaturation step, the Borago officinalis L. Δ 6 desaturase can convert linoleic acid and α-linolenic add to γ-linolenic acid (GLA) and stearidonic acid (STA), respectively. Both GLA and STA are of interest to the pharmaceutical and nutraceutical industries. Production of these fatty acids is costly. One potential strategy to reduce production cost would be to generate them in a major oilseed crop. To this end, a cDNA of the B. officinalis Δ 6 -desaturase gene was cloned downstream of the embryo-specific promoter β-conglycinin. The resultant cassette was assembled into a two T-DNA binary vector, in which the second T-DNA element harbored a selectable marker cassette. The final plasmid was subsequently used to transform soybean [Glycine max (L.) Merr.]. The simultaneous delivery of two T-DNA elements was used as a strategy to derive soybean progeny transgenic for the Δ 6 desaturase T-DNA and free of the marker gene T-DNA. Twenty-nine transgenic soybean lines were recovered that harbored both T-DNA elements, of which 17 produced GLA and STA in the seed storage lipids. Average GLA levels ranged from 3.4 up to 28.7%, while STA levels varied from just under 0.6 to 4.2% in the T 1 generation. Among the 17 lines that produced GLA and STA, four lines were identified that were free of the selectable marker T-DNA element.

For many traits, it is often desirable to transfer multiple genes or to pyramid several novel genes in one line. Understanding and predicting transgene linkage relationships is Transferring multiple genes can be achieved through required to utilize efficiently transformation technologies in breeding programs. This study was conducted to determine the co-transforma- transformation using one gene at a time, but this would tion frequency of three gene sequences by means of a three-plasmid likely prove to be impractical and inefficient (Chen et co-transformation system and to determine the mode of inheritance al., 1998). By means of microprojectile bombardment, for two genes of interest. Twenty-five independently transformed a number of plasmids containing genes of interest and wheat (Triticum aestivum L.) plants were produced by microprojectile a selectable marker can be mixed before bombardment bombardment of 1080 immature embryos with a three-plasmid system. and used for co-transformation (Chen et al., 1998). The Polymerase chain reaction (PCR) analyses of T1 progeny from each co-transformation of multiple transgenes present on of the 25 T0 plants indicated co-transformation of all three plasmids separate plasmids has been employed successfully via at a frequency of 36%. Two of nine transgenic families containing all microprojectile bombardment in a number of plant spethree plasmids were characterized for the segregation of the two genes cies, including common bean (Phaseolus vulgaris L.; of interest in T1 ,T 2, and T2 testcross generations by PCR. These data