Irma Bernal-Lugo

Seed stability during storage: Raffinose content and seed glassy state

It has been proposed that sucrose and raffinose play a role in the storability of maize seeds. The levels of these sugars in the embryos and the glassy state were compared in maize seeds of contrasting storage stability to determine the relationship between sugar composition, glassy state and the storability of the seed. Sucrose was the predominant sugar but its content was not correlated with good storage. The content of raffinose as a mass fraction of total sugars and the magnitude of the glassy state showed positive correlations with storage stability. It is suggested that in maize seeds dry storage stability is a reflection not of the total soluble sugars, but of the mixture of sucrose with raffinose. Apparently raffinose amplifies the magnitude of the glass signal and this in turn is associated with enhanced storability.

Control of superoxide production in mitochondria from maize mesocotyls

To understand the biochemical events that control the generation of superoxide, the effect of inhibiting the respiratory complexes III and IV (C‐III and C‐IV) and alternative oxidase (AOX) on the rate of superoxide production was analyzed in mitochondria from maize seedlings. To increase superoxide production, it was required to inhibit C‐III or C‐IV by at least 30% or 50%, respectively. Below this inhibition threshold, AOX exerted the highest degree of control on superoxide production, whereas above it, the highest degree of control was exerted by C‐IV. The contribution of C‐III to control superoxide production became significant when AOX activity was modulated.

Early carbon mobilization and radicle protrusion in maize germination

Considerable amounts of information is available on the complex carbohydrates that are mobilized and utilized by the seed to support early seedling development. These events occur after radicle has protruded from the seed. However, scarce information is available on the role of the endogenous soluble carbohydrates from the embryo in the first hours of germination. The present work analysed how the soluble carbohydrate reserves in isolated maize embryos are mobilized during 6–24 h of water imbibition, an interval that exclusively embraces the first two phases of the germination process. It was found that sucrose constitutes a very significant reserve in the scutellum and that it is efficiently consumed during the time in which the adjacent embryo axis is engaged in an active metabolism. Sucrose transporter was immunolocalized in the scutellum and in vascular elements. In parallel, a cell-wall invertase activity, which hydrolyses sucrose, developed in the embryo axis, which favoured higher glucose uptake. Sucrose and hexose transporters were active in the embryo tissues, together with the plasma membrane H+-ATPase, which was localized in all embryo regions involved in both nutrient transport and active cell elongation to support radicle extension. It is proposed that, during the initial maize germination phases, a net flow of sucrose takes place from the scutellum towards the embryo axis and regions that undergo elongation. During radicle extension, sucrose and hexose transporters, as well as H+-ATPase, become the fundamental proteins that orchestrate the transport of nutrients required for successful germination.

Enzymatic changes in pectic polysaccharides related to the beneficial effect of soaking on bean cooking time

BACKGROUND Cooking time decreases when beans are soaked first. However, the molecular basis of this decrease remains unclear. To determine the mechanisms involved, changes in both pectic polysaccharides and cell wall enzymes were monitored during soaking. Two cultivars and one breeding line were studied. RESULTS Soaking increased the activity of the cell wall enzymes rhamnogalacturonase, galactanase and polygalacturonase. Their activity in the cell wall was detected as changes in chemical composition of pectic polysaccharides. Rhamnose content decreased but galactose and uronic acid contents increased in the polysaccharides of soaked beans. A decrease in the average molecular weight of the pectin fraction was induced during soaking. The decrease in rhamnose and the polygalacturonase activity were associated (r = 0.933, P = 0.01, and r = 0.725, P = 0.01, respectively) with shorter cooking time after soaking. CONCLUSION Pectic cell wall enzymes are responsible for the changes in rhamnogalacturonan I and polygalacturonan induced during soaking and constitute the biochemical factors that give bean cell walls new polysaccharide arrangements. Rhamnogalacturonan I is dispersed throughout the entire cell wall and interacts with cellulose and hemicellulose fibres, resulting in a higher rate of pectic polysaccharide thermosolubility and, therefore, a shorter cooking time.

Cotyledon thermal behavior and pectic solubility as related to cooking quality in common beans

The characteristic of proteins, starch and pectic substances in cotyledons of two bean cultivars varying in cooking time were determined to investigate their possible contribution to bean cooking quality. Both cultivars showed the same enthalpies of starch gelatinization but different protein denaturation enthalpies. The proportion of hot water soluble pectins was higher in Michigan, the cultivar with the lower cooking time, than in Ojo de Cabra, the cultivar with the higher cooking time. These results were not due to differences in pectin methylation or in the ratio of monovalent to divalent cations in the tissue, suggesting that in fresh beans the β-elimination reaction is not the sole or predominant route of thermal pectin degradation. Overall, this study indicates that varietal differences in bean cooking quality may be reflections of the rate of pectin loss during soaking/heating and that the thermal properties of starch and protein fractions seem to have a minor contribution. Researchers involved in this study propose that in fresh beans, the thermal pectin loss results from a two step mechanism: pectin enzymic breakdown during the bean soaking followed by thermal solubilization rather than β-elimination during the bean heating.

Tonoplast and plasma membrane ATPases from maize lines of high or low vigour.

The effectiveness of ATPase in germinated seed may play an important role in the vigour of germination. The activities of tonoplast and plasma membrane ATPases in two maize ( Zea mays L.) lines with different vigour of germination were determined. ATP hydrolysis was measured in microsomal fractions from coleoptiles along with the responses to specific inhibitors for the plasma membrane, tonoplast and mitochondrial ATPases as well as for acid phosphatase. Nitrate-sensitive ATPase activity was 1.5–3.0 times lower in the low-vigour line than in the high-vigour line. Kinetic analysis of ATP hydrolysis at different substrate concentrations revealed the existence of two enzymes in the microsomal fractions of the two lines. The V max of enzyme 1 in the low-vigour line was a third of that in the high-vigour line. This enzyme was identified as the nitrate-sensitive or tonoplast ATPase on the basis of measurements of ATP hydrolysis in the presence of specific inhibitors at high (8.12m m ) and low (0.77m m ) ATP concentrations.

Effect of pH on the GA3 induced α-amylase synthesis

Summary Isolated wheat aleurone layers acidify the incubation medium from pH 6 to 4, and this acidification is not dependent on GA3 (Hamabata et al., 1988). As an attempt to assesss the physiological role of this acidification, we have examined the effect of pH on the induction of a-amylase by GA3 in isolated wheat aleurone layers (Triticum aestivum L. cv. Tonichi). Both α-amylase production and protein synthesis induced by GA3 showed a strong pH dependence. α-Amylase activity accumulated only at pH below 5. The rate of protein synthesis decreased with the addition of GA3 at pH 6 and 5, compared to that with no GA3 added, while at pH 4 protein synthesis induced by GA3 increased almost twice. At pH 5 and 6 the SDS-PAGE fluorography of aleurone total proteins did not change significatively by GA3 addition. However, at pH 4, GA3 caused the appearance of a predominant band with the same relative molecular weight as that of purified α-amylase. The above results strongly suggest that GA3 has no action unless the aleurone layer acidifies its surrondings. In this sense, the acidification caused by aleurone layers has a permissive control on the physiological actions of GA3.

Resistance of wheat aleurone cell walls to acid and xylanase action

Summary Isolated wheat (Triticum aestivum var. Potam) aleurone layers have a high capacity to acidify their environment, and secrete hydrolytic enzymes (endoxylanase, glucanase, α-amylase, proteases, etc.) under the control of GA3. Acidic pH and xylanases are found to be essential for cell wall relaxation in growing tissues, but aleurone is a non-growing, non-dividing tissue. In this tissue, we studied the effect of these loosening factors on aleurone cell walls.Exposure to pH 3.0 caused the release of carbohydrates and calcium ions from the pericarp, and a small amount of carbohydrates, mainly polysaccharides, from aleurone layers from which pericarp tissue had been removed. 50 percnt; of the total sugars released into the incubation medium by these isolated aleurone tissue was arabinose, but no xylose, calcium ions, or phenolic compounds were found. Acid preincubation decreased by 30 percnt; the susceptibility of aleurone cell walls to degradation by exogenously-applied endoxylanase, and also modified the architecture of cell wall as observed by autofluorescence of phenolic groups. These findings suggest that acid treatment and endoxylanase action, rather than having a loosening effect on aleurone cell wall, can have an opposite effect, increasing the resistance of aleurone cell walls to loosening.

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.