Ribosomal Heterogeneity of Maize Tissues: Insights of Biological Relevance

In recent years, the selective role of ribosomes in the translational process of eukaryotes has been suggested. Evidence indicates that ribosomal heterogeneity at the level of protein stoichiometry and phosphorylation status differs among organisms, suggesting ribosomal specialization according to the state of development and the surrounding environment. During germination, protein synthesis is an active process that begins with the translation of the mRNAs stored in quiescent seeds and continues with the newly synthesized mRNAs. In this study, we identified differences in the abundance of ribosomal proteins (RPs) in maize embryos at different developmental stages. The relative quantification of RPs during germination revealed changes in six small subunit proteins, S3 (uS3), S5 (uS7), S7 (eS7), two isoforms of S17 (eS17), and S18 (uS13), and nine large subunit proteins, L1 (uL1), L5 (uL18), two isoforms of P0 (uL10), L11 (uL5), L14 (eL14), L15 (eL15), L19 (eL19), and L27 (eL27). Further analysis of ribosomal protein phosphorylation during germination revealed that the phosphorylation of PRP0 (uL10) and P1 increased and that of PRS3 (uS3) decreased in germinated versus quiescent embryos. The addition of insulin during germination increased the phosphorylation of the P1 protein, suggesting that its phosphorylation is controlled by the TOR pathway. Our results indicate that a heterogeneous ribosomal population provides to maize ribosomes during germination a different ability to translate mRNAs, suggesting another level of regulation by the ribosomes.     

The families of papain- and legumain-like cysteine proteinases from embryonic axes and cotyledons of Vicia seeds: developmental patterns,intracellular localization and functions in globulin proteolysis

Families of papain- and legumain-like cysteine proteinases (CPR) were found in Vicia seeds. cDNAs and antibodies were used to follow organ specificity and the developmental course of CPR-specific mRNAs and polypeptides. Four papain-like cysteine proteinases (CPR1, CPR2, proteinase A and CPR4) from vetch seeds (Vicia sativa L.) were analysed. CPR2 and its mRNA were already found in dry embryonic axes. CPR1 was only detected there during early germination. Both CPR1 and CPR2 strongly increased later during germination. In cotyledons, both CPR1 and CPR2 were only observed one to two days later than in the axis. Proteinase A was not found in axes. In cotyledons it could only be detected several days after seeds had germinated. CPR4 mRNA and polypeptide were already present in embryonic axes and cotyledons during seed maturation and decreased in both organs during germination. Purified CPR1, CPR2 and proteinase A exhibited partially different patterns of globulin degradation products in vitro. Although the cDNA-deduced amino acid sequence of the precursor of proteinase A has an N-terminal signal peptide, the enzyme was not found in vacuoles whereas the other papain-like CPRs showed vacuolar localization. Four different legumain-like cysteine proteinases (VsPB2, proteinase B, VnPB1 and VnPB2) of Vicia species were analysed. Proteinase B and VnPB1 mRNAs were detected in cotyledons and seedling organs after seeds had germinated. Proteinase B degraded globulins isolated from mature vetch seeds in vitro. VsPB2 and proteinase B are localized to protein bodies of maturing seeds and seedlings, respectively, of V. sativa. Like VsPB2 from V. sativa, also VnPB2 of V. narbonensis corresponds to vacuolar processing enzymes (VPE). Based on these results different functions in molecular maturation and mobilization of storage proteins could be attributed to the various members of the CPR families.     

Maize DNA polymerase alpha is phosphorylated by a PCNA-associated cyclin/Cdk complex: effect of benzyladenine

The activity of maize DNA polymerases 1 and 2 (delta and alpha-type enzymes, respectively) is stimulated during germination if embryo axes are imbibed in the presence of benzyladenine. In vivo, DNA pol 2 is a phosphorotein that appears to be maximally phosphorylated previous to the S phase start time (by 12 h of germination, Coello and Vázquez-Ramos 1995a). We find that, in vitro, a PCNA-associated cyclin/kinase activity isolated from maize axes acquires an increasing capacity to phosphorylate DNA pol 2 as germination advances; moreover, the PCNA-associated kinase isolated from BA-treated maize axes germinated at 3 h phosphorylates DNA pol 2 at the same level observed in samples of axes germinated for 13 h in the absence of exogenous BA. PCNA-associated kinase activity from BA-treated axes germinated at 13 h maximal using DNA pol 2 as substrate. However, there is no modification in DNA polymerase activity as a consequence of protein phosphorylation. Results are discussed in terms of their significance for cell cycle regulation during seed germination.     

Spatially regulated genes expressed during seed germination and postgerminative development are activated during embryogeny

Summary We investigated the control of genes expressed primarily during seed germination and postgerminative development in Brassica napus L. We identified cloned mRNA sequences which became prevalent within 1 day after the start of imbibition and were at low or undetectable levels in immature embryos, dry seeds, and leaves. Most postgermination-abundant mRNAs accumulated primarily, though not exclusively, in different parts of the seedling. Of the 14 cloned mRNAs, 8 were prevalent in cotyledons, 2 were abundant in seedling axes, and 4 were approximately equally distributed in both parts. We showed that although these mRNAs reached maximal levels in seedlings, the spatially regulated mRNAs were also detected at distinct embryonic stages; mRNAs prevalent in seedling axes accumulated primarily during early embryogenesis while cotyledon-abundant mRNA concentration increased during late embryogeny. We conclude that the temporal and spatial regulation of gene expression in seedlings reflects similarities and differences in the physiological functions of cotyledons and axes. Furthermore, the regulated expression of cotyledon-abundant genes during late embryogeny suggests that the mRNAs and possibly proteins may accumulate in preparation for subsequent seedling growth. Similarities in the accumulation of cotyledon-abundant mRNAs may indicate coordinate regulation of this gene set.Abbreviations DAF days after flowering - DAI days after the start of imbibition - HAI hours after the start of imbibition - kb kilobase(pairs)     

Stored proteinases and the initiation of storage protein mobilization in seeds during germination and seedling growth

Though endopeptidases and carboxypeptidases are present in protein bodies of dry quiescent seeds the function of these proteases during germination is still a matter of debate. In some plants it was demonstrated that endopeptidases of dry protein bodies degrade storage proteins of these organelles. Other studies describe cases where this did not happen. The role that stored proteinases play in the initiation of storage protein breakdown in germinating seeds thus remains unclear. Numerous reviews state that the initiation of reserve protein mobilization is attributed to de novo formed endopeptidases which together with stored carboxypeptidases degrade the bulk of proteins in storage organs and tissues after seeds have germinated. The evidence that the small amounts of endopeptidases in protein bodies of embryonic axes and cotyledons of dry seeds from dicotyledonous plants play an important role in the initiation of storage protein mobilization during early germination is summarized here.     

Acquisition of desiccation tolerance in soybeans

The entry into a desiccation-tolerant state is a major developmental component of seed maturation. Development of desiccation tolerance of embryonic axes of soybean [Glycine max (L.) Merrill cv. Chippewa 64] was studied by measuring changes in electrolyte leakage. germination and relative growth rate after axes were rapidly air-dried to various water contents. Axes acquired the full capacity for germination at 34 days after flowering (DAF). and reached physiological maturity (maximum dry weight) at 48 DAF. When dried to water content h = 0. 08 (g water g⁻¹ dry weight). few axes germinated before 42 DAF. but more than 90% germinated after 48 DAF. However, electrolyte leakage of rehydrated axes showed a linear decline from 30 to 55 DAF. For developing axes there was a critical water content or desiccation threshold. which could be estimated by using the electrolyte leakage method. The threshold of desiccation tolerance decreased gradually from h = 1. 10 to 0. 18 as axes matured from 28 to 55 DAF. The development of desiccation tolerance continued after physiological maturity at 48 DAF. We conclude that the acquisition of desiccation tolerance of soybean axes is a gradual event, rather than an abrupt transition.     

Expression of three ABA-regulated clones and their relationship to maturation processes during the embryogenesis of chick-pea seeds

Water stress inhibits germination of chick-pea seeds and produces specific changes in gene expression. some of which are coincident with those induced by the exogenous application of abscisic acid (ABA). Three cDNA clones, GAB-8, GAB-9 and GAB-11, were previously identified as under the regulation of ABA and osmotic stress in embryonic axes of germinating chick-pea. Here we try to establish a relationship between the changes in gene expression induced by ABA and stress conditions during germination and those naturally occurring during the desiccation process that leads to seed maturation. Our results show that the germinative capacity of chick-pea is related to the water content of the organ. In vitro translation of the mRNAs from developing seed reveals that in the later stages of seed maturation some polypeptides appear that previously were found to be regulated by ABA and by water deficit in germinating seeds. Hybridization by northern blot of embryogenic mRNAs with GAB-8. GAB-9 and GAB-11 clones shows that the mRNAs corresponding to such clones only appear in the later phases of seed formation, coinciding with seed dehydration, and persisting until seeds became fully mature. The results suggest that these mRNAs are probably related to the response to dehydration that occurs during seed maturation, and that the pattern of expression of these ABA-regulated clones coincides with that of the established late embryogenesis-abundant (LEA) genes.     

Abscisic acid and temperature modify the levels of calmodulin in embryonic axes of Cicer arietinum

Changes in calmodulin (CaM) levels in embryonic axes of Cicer arietinum L. cv. Castellana germinated under three different conditions were measured. Abscisic acid (ABA) and a temperature of 30°C, which delay chick-pea germination, respectively decreased and increased the concentration of CaM compared to the values obtained under normal germinating conditions (H₂O-25°C). The CaM concentration was higher in those zones of the axes undergoing an active cell division.
The compartmentalization of CaM in 36-h-old embryonic axes grown under these three conditions was also measured. Cytosolic and mitochondrial CaM was higher in axes where a delayed germination occurred as well as in the cell walls of normally grown ones. On the other hand, CaM was higher in nuclear and microsomal fractions extracted from H₂-O-25°C-treated axes. From these data we postulate that delayed germination could be an effect of altered CaM distribution in chick-pea embryonic axes.     

Influence of the Embryonic Axis on Protein Hydrolysis in Cotyledons of Cucurbita maxima

Four-day time course studies of the hydrolysis of cotyledonal storage protein were conducted on intact seeds, seed cotyledons detached from their embryonic axes and on detached cotyledon pairs germinated in the presence of three excised embryonic axes of Cucurbita maxima Duch., cv. Chicago Worted Hubbard. Detached cotyledons germinated alone showed little hydrolysis of the storage protein. However, the amount of protein hydrolysis of the detached cotyledon pairs germinated in the presence of three excised embryonic axes was comparable to the amount hydrolyzed in the cotyledons of intact germinating seeds. Visual growth differences among these treatments were also evident. The size and yellow color intensity of the fourth day treatments were shown to increase in the following order: detached cotyledon pairs alone, intact seedlings, detached cotyledon pairs in the presence of three excised axes. The growth of the hypocotyl and radical was also modified by removal of the cotyledons. These findings suggest that storage protein degradation and cotyledonal growth are controled by the axis. They also indicate that the cotyledons have some influence on the growth of the axes. Time-course studies were made on the hydrolysis of storage protein in the cotyledons of squash and on the distribution of the hydrolytic products during the germination of light- and dark-grown plants. The storage protein was not hydrolyzed during the first 24 hours. It was hydrolyzed at a uniform rate from 1 to 5 days and at a slightly decreased rate from 5 to 7 days. Most of the hydrolytic products were transported to the axial tissue. Proteinase activity in the cotyledons rapidly increased during germination to a maximum level at 2 to 3 days. This was followed by a decline to about the initial value after 7 days.