Carlo Soave

Heterogeneity of storage proteins in maize

The extensive charge heterogeneity of maize (Zea mays L.) zeins observed in isoelectric focusing (IEF) (about 15 bands with pIs in the pH range 6–9) has been found to be independent of extraction procedures or of endosperm development. Zeins do not stain for glycoproteins and exhibit only one lipoprotein component, with pI 3, representing 3–5% of the total protein.Zeins are very resistant to in vitro deamidation, at both acidic and alkaline pH, at high temperatures, and for rather prolonged times. On the basis of the zein content in acidic and basic amino acids, and of the respective pIs exhibited in IEF (mostly in the pH range 7–8) it has been calculated that at least 90% of the glutamic and aspartic acids (≈52 residues out of a total of ≈ 190) are present as asparagine and glutamine.Amino acid analysis of zein fractions isolated by preparative IEF has demonstrated changes in the composition of 18 amino acid residues. However, since these changes affect only neutral and hydrophobic residues, it is concluded that the observed zein heterogeneity is partly based on in vivo deamidation of glutamine and asparagine and partly to spot mutations in some of the genes responsible for zein synthesis.

Amino acid composition of zein molecular components

Abstract Zein extracted from maize endosperm has been fractionated into four polypeptide chains, having the following MWs 23 000, 21 000, 13 500 and 9600. By amino acid analysis the two smaller MW chains (representing 30% of total zeins) have been found to be zein-type molecules. These two chains are thought to be responsible for zein granule formation via -S-S- bridges. Zein is also highly heterogeneous in charge, and is resolved into at least 15 components, with pIs in the pH range 5–9. As demonstrated by amino acid analysis, part of this heterogeneity is due to spot mutations in some of the genes responsible for zein synthesis.

Molecular cloning of the o2-m5 allele of Zea mays using transposon marking

SummaryThe deposition of zein protein in maize endosperm is under the control of several regulatory loci. The isolation of DNA sequences corresponding to Opaque-2 (O2), one of such loci, is described in this paper. The mutable allele, o2-m5 was first induced moving the Ac transposable element present at the wx-m7 allele to the O2 locus. Genetic data suggest that a functional Ac element is responsible for the observed somatic mutability of o2-m5. The isolation of genomic clones containing flanking sequences corresponding to the O2 gene was possible by screening an o2-m5 genomic libary with a probe corresponding to internal Ac sequences usually absent in the defective element Ds. Out of 27 clones isolated with homology to the central part of Ac element, only clones 6IP and 21IP generated a 2.5 kb internal fragment size of an active Ac element when digested with PvuII restriction enzyme. A sequence representing a XhoI fragment of 0.9 kb lying, in the 6IP clone, adjacent to the Ac elements, was subcloned and utilized to prove that it corresponded to a part of the O2 gene. To obtain this information we made use of: (1) DNAs from several reversions originating from the unstable (o2mk-(r) allele, which, when digested with SstI, showed a correct 3.4 kb fragment typical of non-inserted alleles of the O2 locus; and (2) recessive alleles of the O2 locus which were devoid of a 2.0 kb mRNA, present on the contrary in the wild type and in other zein regulating mutants different from O2.

Suppression of Both ELIP1 and ELIP2 in Arabidopsis Does Not Affect Tolerance to Photoinhibition and Photooxidative Stress

ELIPs (early light-induced proteins) are thylakoid proteins transiently induced during greening of etiolated seedlings and during exposure to high light stress conditions. This expression pattern suggests that these proteins may be involved in the protection of the photosynthetic apparatus against photooxidative damage. To test this hypothesis, we have generated Arabidopsis (Arabidopsis thaliana) mutant plants null for both elip genes (Elip1 and Elip2) and have analyzed their sensitivity to light during greening of seedlings and to high light and cold in mature plants. In particular, we have evaluated the extent of damage to photosystem II, the level of lipid peroxidation, the presence of uncoupled chlorophyll molecules, and the nonphotochemical quenching of excitation energy. The absence of ELIPs during greening at moderate light intensities slightly reduced the rate of chlorophyll accumulation but did not modify the extent of photoinhibition. In mature plants, the absence of ELIP1 and ELIP2 did not modify the sensitivity to photoinhibition and photooxidation or the ability to recover from light stress. This raises questions about the photoprotective function of these proteins. Moreover, no compensatory accumulation of other ELIP-like proteins (SEPs, OHPs) was found in the elip1/elip2 double mutant during high light stress. elip1/elip2 mutant plants show only a slight reduction in the chlorophyll content in mature leaves and greening seedlings and a lower zeaxanthin accumulation in high light conditions, suggesting that ELIPs could somehow affect the stability or synthesis of these pigments. On the basis of these results, we make a number of suggestions concerning the biological function of ELIPs.

Zein level in Maize endosperm depends on a protein under control of the opaque-2 and opaque-6 loci

Zeins, the major endosperm proteins of maize, represent about 50% of total seed proteins and consist of several alcohol-soluble polypeptides encoded by at least four families of genes. The accumulation of zeins is under the control of several broadly defined regulatory genes. One of these, the opaque-2 mutant, lowers the level of zeins, thus improving the nutritional quality of maize meals. We demonstrate here that a salt-soluble protein of molecular weight 32,000 (b-32) is under control of O2. Seven o2 recessive alleles are CRM- when assayed with b-32 antibody. The O2 gene does not encode protein b-32, which apparently is the gene product of the O6 locus. 06 endosperms are CRM- for b-32 protein and are almost devoid of zeins. This indicates that b-32 plays an important role in accumulation of zeins.

Transport of amino acids with special emphasis on the synthesis and transport of asparagine in the Illinois Low Protein and Illinois High Protein strains of maize

Abstract. In order to understand the metabolic processes governing the protein content in maize grains, we compared metabolic parameters of the two maize strains Illinois High Protein (IHP) and Illinois Low Protein (ILP), which differ largely in their relative starch and protein content. The activity of nitrate reductase (NR) and the transcription level of asparagine synthetase, two enzymes that play a central role in the flow of N into organic compounds, were also investigated. It was shown that IHP plants contained a higher activity of NR than ILP plants, which was in part due to an increased level of NR activity in the roots. The root-to-shoot ratios in the ILP and IHP strains were 0.41 and 0.54, respectively. The total amino nitrogen contained in the amino acids of the leaves, phloem, xylem and grain was much higher in IHP plants than in ILP plants. The enhanced contents of amino nitrogen in IHP plants were mainly due to an increase in the asparagine level, which was 13 times higher in the xylem of IHP plants than in that of ILP plants. In the roots the asparagine synthetase was expressed constitutively. We conclude from these results that IHP maize differs from ILP maize in having a high capacity for delivering asparagine as a product of root metabolism in addition to the amino acids supplied by nitrate assimilation of the leaves.

Antisense reduction of thylakoidal ascorbate peroxidase in Arabidopsis enhances Paraquat-induced photooxidative stress and Nitric Oxide-induced cell death

The production and characterization of Arabidopsis plants containing a transgene in which the Arabidopsis tAPX is inserted in antisense orientation, is described. tAPX activity in these transgenic tAPX plants is around 50% of control level. The tAPX antisense plants are phenotypically indistinguishable from control plants under normal growth conditions; they show, however, enhanced sensitivity to the O2−-generating herbicide, Paraquat. Interestingly, the tAPX antisense plants show enhanced symptoms of damage when cell death is triggered through treatment with the nitric oxide-donor, SNP. These results are in accordance with the ones recently obtained with transgenic plants overexpressing tAPX; altogether, they suggest that tAPX, besides the known ROS scavenging role, is also involved in the fine changes of H2O2 concentration during signaling events.

Preparation and functional characterization of thylakoids from Arabidopsis thaliana

A protocol for the isolation of functional thylakoids from Arabidopsis thaliana leaves was developed. The critical factor in obtaining active, coupled and stable preparation is the inclusion of EDTA and EGTA in the grinding buffer. Preparations were characterized with respect to the whole or partial electron transport chain, ATP/NADPH, ATP/O2 and PS II/chlorophyll ratios. Sensitivity to a light-chill photoinhibitory treatment was also determined by evaluating the decrease in both maximal photochemical efficiency (Fv/Fm) and in electron transport rate.

Differential involvement of the IDRS cis-element in the developmental and environmental regulation of the AtFer1 ferritin gene from Arabidopsis

Four different ferritin genes have been identified in Arabidopsis thaliana, namely AtFer1, 2, 3 and 4. AtFer1, which strongly accumulates in leaves treated with excess iron, contains in its promoter an Iron-Dependent Regulatory Sequence (IDRS). The IDRS sequence is responsible for repression of AtFer1 transcription under conditions of low iron supply. Arabidopsis plants transformed with a 1,400-bp AtFer1 promoter, with either a wild-type or a mutated IDRS fused to the β-glucuronidase (GUS) reporter gene, enabled us to analyze the activity of the AtFer1 promoter in different tissues as well as during age-dependent or dark-induced senescence. Our results show that IDRS mediates AtFer1 expression during dark-induced senescence while it does not affect AtFer1 expression during age-dependent senescence or in young seedlings. Photoinhibition promoted either by high light or chilling temperature, or wounding, does not activate the AtFer1 promoter. In contrast, AtFer2, AtFer3, AtFer4 transcript abundances are increased in response to photoinhibition and AtFer3 transcript abundance is increased upon wounding. Taken together, our results indicate that other cis-elements, different from the IDRS, regulate the territory-specific or developmental expression of AtFer1 gene. Expression of this gene appears insensitive to some of the environmental stresses tested, which instead up-regulate other members of the Arabidopsis ferritin gene family.

Knockout of frataxin gene causes embryo lethality in Arabidopsis

The frataxin homolog from Arabidopsis thaliana (AtFH) is a single nuclear‐encoded gene targeted to mitochondria and sharing 65% similarity with animal frataxin. In the present work, we show that the knocking out of AtFH gene causes arrest of Arabidopsis embryo development at the globular stage. Consistently with that, we also show by in situ hybridization that AtFH is expressed, in wt Arabidopsis plants, in ovule primordia as well as in embryos at various stages of development, suggesting a key role of plant frataxin during embryogenesis.