The suppression of the glutelin storage protein gene in transgenic rice seeds results in a higher yield of recombinant protein

Glutelin is a major seed storage protein, accounting for 60?C80?% of the total endosperm protein content in rice. To test whether we could augment the expression of an introduced recombinant protein in rice by suppressing the glutelin gene, we generated transgenic glutelin RNAi (glu RNAi) rice seeds. RNA gel blot analyses confirmed that the endogenous glutelin gene was severely suppressed in these transgenic rice lines. RT-PCR analysis further revealed that all the members of glutelin multigene family were downregulated. Transgenic glu RNAi rice seeds expressing a recombinant red fluorescent protein (RFP) showed stronger fluorescence than seeds transformed with the RFP gene only. Western blot analysis further revealed that the relative accumulation of RFP in glu RNAi seeds was twofold higher than that in the RFP-only transgenic seeds. These results suggest that RNAi targeting of an endogenous storage protein could be of great utility in obtaining higher transgene expression in genetically engineered rice and other plant lines.     

Glutelin accumulation and changes in the levels of its mRNA in the superior and inferior spikelets of rice ear during ripening

Glutelin accumulation in the apical spikelet of the top primary branch (superior spikelet) and the second spikelet of the lowest secondary branch (inferior spikelet) of the ear of the rice plant (Oryza sativa L.) was characterized during grain filling.In the superior spikelet, the accumulation of dry matter and nitrogen started immediately after flowering and rapidly reached the maturation level by 20 days after heading (DAH). At 7 DAH, total RNA content had already reached its maximum level and glutelin mRNA content 70% of its maximum. The increase in glutelin mRNA was followed by a rapid increase in glutelin between 7 and 16 DAH.In the inferior spikelet dry matter, nitrogen and glutelin accumulation were low immediately after flowering and increased only after grain filling of the superior spikelet was almost complete. Total RNA and glutelin mRNA increased much later at slower rates than in the superior spikelet.It is very likely that the retardation of dry matter, total nitrogen and glutelin accumulation in the inferior spikelet is due to retardation of differentiation and development of endosperm tissue, and to glutelin gene expression in endosperm cells. It is suggested that the delayed development resulted from limited partitioning of nutrients to the inferior spikelet at the early stage of ripening.     

Development of specific rRNA probes to distinguish between geographic clades of the Alexandrium tamarense species complex

The globally occurring Alexandrium tamarense/fundyense/catenellaspecies complex consists of toxic and non-toxic strains thatare morphologically difficult to distinguish. We developed fourspecific ribosomal RNA probes that can identify the entire speciescomplex, the strains of the toxic North American clade and thestrains of the two non-toxic clades from Western Europe andthe Mediterranean Sea by DNA dot blot and fluorescence in situhybridization. These probes are a first step for the developmentof an early warning system for the presence of A. tamarense.     

Changes in nitrogen contents and in proteolytic activities in different parts of field-grown wheat ears (Triticum aestivum L.) during maturation

Aminopeptidase, carboxypeptidase and neutral endopeptidase activitieswere analyzed in glumes and in kernels of field-grown wheat(Triticum aestivum L.) during ear development. Kernels harvestedon two dates were subdivided into outer pericarp, cross cells,endosperm and embryo. In developing parts with a net nitrogeninflux (young glumes, embryo, endosperm) the aminopeptidaseactivity is high, but in nitrogen-mobilizing tissues (senescingglumes, Outer pericarp) this activity decreases. Carboxypeptidaseis most active in fully expanded tissues. Neutral endopeptidaseshows the highest activity in the nitrogen mobilizing partsand extremely low activity in the embryo and the endosperm. (Received July 15, 1978; )     

CHANGES IN THE CARBOHYDRATES, PROTEINS AND NUCLEIC ACIDS IN THE OVULES OF ZEPHYRANTHES LANCASTERI AT DIFFERENT STAGES OF MATURATION

The major changes in the levels of soluble and insoluble carbohydrates,nitrogenous compounds and nucleic acids were investigated atdifferent stages of seed development in Zephyranthes lancasteri.The activities of amylases, glutamic-alanine transaminase, ribonucleaseand deoxyribonuclease were also studied. The alcohol-solublenitrogen and carbohydrates attain their maximal levels priorto the elongation ofthe cotyledon. Both of these decrease markedlyduring further maturation of the seed. The accumulation of totalnitrogen in the ovule follows a sigmoid pattern. The glutamic-alaninetransaminase activity appears to be exclusively localized inthe endosperm and is absent from the embryo. The embryo seemsto derive its organic nitrogen fromthe endosperm. The peak inthe level of DNA per ovule is attainedprior to the elongationof the cotyledon while that of RNA is foundsoon after. ¹Present address: AEC Plant Research Laboratory, Michigan StateUniversity, East Lansing, Mich., U.S.A.     

Tissue-Dependent Expression of the Rice wx+ Gene Promoter in Transgenic Rice and Petunia

The waxy (wx) locus, which controls the amylose synthesis, isknown to be expressed specifically in the endosperm and pollen.To study the tissue-specific regulation of the wx⁺ gene, weintroduced a fusion gene that consisted of the upstream sequenceof the wx⁺ gene and the gene for rß-glucuronidase(GUS) into cells of rice (Oryza sativa L.) and petunia (Petuniahybrida L.). GUS activity was examined in the regenerated transgenicrice and petunia plants. In transgenic rice, the upstream sequenceof the wx⁺ gene was sufficient to direct the tissue-specificexpression of GUS in the endosperm and pollen, and the controlof expression was quantitative. By contrast, in transgenic petunia,the same fusion gene was expressed in pollen but not in theendosperm. These results suggest that the putative cis-actingelements that direct pollen-specific expression are common toor similar in both monocotyledonous and dicotyledonous plants,whereas ciy-elements responsible for the endosperm-specificexpression of the rice wx⁺ gene do not function in petunia,in which development of the endosperm differs from that in rice. ⁴Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan     

Development of the Almond Nut (Prunus dulcis (Mill.) D. A. Webb). Anatomy and Chemical Composition of Fruit Parts from Anthesis to Maturity

The growth of the fruit of two varieties of almond (Prunus dulcis(Mill.) D. A. Webb) was studid from anthesis (week 0) to maturity(week 32). The dimensions, fresh weight, moisture content, anatomyand chemical composition of the pericarp, testa, embryo, endospermand nucellus are recorded diagrarnmatically, graphically andby micrographs for one variety. Of the two ovules present atflowering only one normally developed further. By 12 weeks afterflowering the whole fruit had reached full size. The space encloscdby the pericarp was filled by nuallus until weck 10, with subsequentenlargement of both endosperm and embryo. From week 16 to week20 the embryo increased to full size with a concumnt decreasein the size of the endosperm. Sixteen weeks after flowering,the embryo began to accumulate protein and lipid, little ofwhich originated from either the nucellus or endosperm. Theembryo contained no starch or reducing sugar but up to 3% sucrosein the early stags which dtcreascd as lipid and protein increased.Starch and sucrose levels were high in the testa at week 16but subsbquently dropped, starch more rapidly than sucrose.The role of the testa in transport of metabolites to the embryois discussed. Prunus dulcis, almond, fruit development, anatomy, embryo, endosperm     

Normalizing Development of Cultured Triticum aestivum L. Embryos: I. LOW OXYGEN TENSIONS AND EXOGENOUS ABA

Wheat (Triticum aestivum L.) embryos form in dynamically-regulatedovular environments. Our objectives were to improve developmentof cultured immature wheat embryos by simulating, in vitro,abscisic acid (ABA) levels and O₂ tensions as found in wheatovules during zygotic embryogenesis. We characterized from intactwheat kernels embryo respiration, embryo morphology and embryoand endosperm + ABA levels at 13, 19 and 25 d post-anthesis(DPA). Young (13 DPA) embryos were then excised and culturedin vitro, where they were exposed to 0·2 or 2·Ommol m^–3 ±ABA and 2.·1, 2·5 or 7·4mol m^–3 (6, 7 and 21%, respectively) gaseous O₂. At 6and 12 d in culture, + ABA levels, embryo respiration and embryomorphology were characterized by treatment. Thirteen-day-oldembryos from two different plant populations differed by 17-foldin initial ABA content. However, this difference did not affectprecocious germination in vitro, nor did it affect the amountof exogenous ABA required to reduce precocious germination by40%. In this respect, embryos from both populations were equallysensitive to exogenous ABA. Cavity sap O₂ levels (2·1to 2·5 mol m^–3) were much more effective in preventingprecocious germination of cultured embryos than were cavitysap levels of ABA (0·2 to 2·0 mmol m^–3).The combination of physiological levels of both ABA and O₂ largelynormalized DW accumulation and embryo morphology without alteringendogenous + ABA levels. Residual respiration of cultured embryoswas higher than that of embryos grown in situ, and was not influencedby the exogenous O₂ and ABA treatments Key words: Abscisic acid, embryo development, oxygen tensions, respiration, wheat