Polyclonal antibodies to purified recombinant barley haemoglobin (Hb) have been raised in rabbits and used to investigate its expression in monocotyledonous plants. Very little or no Hb expression was observed in dry barley seeds but germination resulted in the expression of Hb which peaked at 2–3 days after imbibition. Hb expression was also observed in maize, wheat, wild oat andEchinochloa crus-galliseeds during germination. Dissection of tissues from the barley seedlings showed that most of the haemoglobin was expressed in the root and seed coat (aleurone layer), with very little in the coleoptile. Imbibition of half-seeds or excised embryos resulted in the expression of haemoglobin. ATP measurements of barley embryos showed that ATP levels quickly increase after imbibition. α-Amylase activity was also determined in embryos to correlate Hb expression with a well-characterized germination response. The results demonstrate that Hb expression is a normal consequence of germination.

Promotion of the albumin storage protein gene response had a more strict stereochemical requirement for elicitation of an ABA response than inhibition of a-amylase gene expression. l h e naturally occurring stereoisomer of ABA and its metabolites were more effective at followed by 7'-hydroxyABA, with phaseic acid being the least active. Racemic 8'-hydroxy-2',3'-dihydroABA, an analog of 8'-hydroxyABA, was inactive, whereas racemic 2',3'-dihydroABA was as effective as ABA. The differences in response of the same tissue to the ABA enantiomers lead us to conclude that there exists more than one type of ABA receptor and/or multiple signal transduction pathways in barley aleurone tissue.

The effects of exogenous phaseic acid (PA) on germination and protein accumulation of cultured immature barley embryos were examined. Chemically synthesized PA was racemic, 87% pure and stable over the course of the experiment. Germination was observable in >90% of the untreated embryos after 3 days of incubation, whereas embryos treated with 10μm abscisic acid (ABA) or PA showed no evidence of germination. Buffer extracts from embryos treated with ABA or PA had similar protein profiles when examined by single and two-dimensional electrophoresis. The profiles differed significantly from those of embryos incubated in the absence of the two compounds. Concentrations of α-amylase inhibitor and barley-germ agglutinin (BGA) increased upon treatment of immature embryos with ABA or PA. This was due to de novo synthesis as there was increased incorporation of radioactivity from 35S-labelled amino acids into the proteins in treated embryos. Endogenous ABA content in PA-treated embryos was not significantly different from that in untreated embryos. An analogue of ABA, 2′, 3′-dihydroabscisic acid, which cannot be metabolized to phaseic acid, inhibited germination and caused increased synthesis of α-amylase inhibitor and germ agglutinin. ABA and PA may both be active in promoting responses associated with ABA in barley embryos, but the embryos are more sensitive to ABA.

Hemoglobin gene expression is upregulated during hypoxia. To determine whether the induction occurs via similar mechanisms that have been proposed for other hypoxically induced proteins, barley (Hordeum vulgare L.) aleurone layers were treated with various agents that interfere with known components of signal transduction. Ruthenium red, an organelle calcium channel blocker, inhibited anoxia-induced hemoglobin (Hb) and alcohol dehydrogenase (EC 1.1.1.1) (Adh) gene expression in a dose-dependent manner. The divalent ionophore, A23187, combined with EGTA also dramatically reduced anoxia-induced Hb and Adh expression. Normal induction of Hb by anoxia in EGTA-treated cells was restored by adding exogenous Ca2+ but not Mg2+, suggesting that cytosolic calcium is involved in Hb and Adh regulation. W-7, a calmodulin antagonist, did not affect anaerobically induced Hb and Adh expression even though it induced Hb under aerobiosis. A3, a protein kinase inhibitor, did not significantly affect anaerobically induced Hb, but did significantly upregulate the gene under aerobic conditions. The results indicate that calmodulin-independent anaerobic alteration in cytosolic Ca2+ and protein dephosphorylation are factors in Hb induction.

SHOOTMERISTEMLESS (STM) is a homeobox gene conserved among plant species which is required for the formation and maintenance of the shoot meristem by suppressing differentiation and maintaining an undetermined cell fate within the apical pole. To assess further the role of this gene during seed storage accumulation, transgenic Brassica napus (Bn) plants overexpressing or down-regulating BnSTM under the control of the 35S promoter were generated. Overexpression of BnSTM increased seed oil content without affecting the protein and sucrose level. These changes were accompanied by the induction of genes encoding several transcription factors promoting fatty acid (FA) synthesis: LEAFY COTYLEDON1 (BnLEC1), BnLEC2, and WRINKLE1 (BnWRI1). In addition, expression of key representative enzymes involved in sucrose metabolism, glycolysis, and FA biosynthesis was up-regulated in developing seeds ectopically expressing BnSTM. These distinctive expression patterns support the view of an increased carbon flux to the FA biosynthetic pathway in developing transformed seeds. The overexpression of BnSTM also resulted in a desirable reduction of seed glucosinolate (GLS) levels ascribed to a transcriptional repression of key enzymes participating in the GLS biosynthetic pathway, and possibly to the differential utilization of common precursors for GLS and indole-3-acetic acid synthesis. No changes in oil and GLS levels were observed in lines down-regulating BnSTM. Taken together, these findings provide evidence for a novel function for BnSTM in promoting desirable changes in seed oil and GLS levels when overexpressed in B. napus plants, and demonstrate that this gene can be used as a target for genetic improvement of oilseed species.