Dolors Ludevid

LYSINE-RICH MODIFIED GAMMA -ZEINS ACCUMULATE IN PROTEIN BODIES OF TRANSIENTLY TRANSFORMED MAIZE ENDOSPERMS

During maize seed development, endosperm cells synthesize large amounts of storage proteins, α-, β, and γ-zeins, which accumulate within endoplasmic reticulum (ER)-derived protein bodies. The absence of lysine in all zein polypeptides results in an imbalance in the amino acid composition of maize seeds. We modified the maize γ-zein gene through the introduction of lysine-rich (Pro-Lys)n coding sequences at different sites of the γ-zein coding sequence. Maize endosperms were transiently transformed by biolistic bombardment with Lys-rich γ-zein constructs under the control of the 1.7 kb γ-zein seed-specific promoter and the cauliflower mosaic virus (CaMV) 35S promoter. When (Pro-Lys)n sequences were inserted contiguous to or in substitution of the Pro-Xaa region of the γ-zein, high levels of protein were observed. In contrast, when (Pro-Lys)n sequences were inserted five residues from the C-terminal, the transcript was present but modified protein was not detected. These results suggest that only an appropriate positioning of Lys-rich inserts leads to the modified molecule displaying correct folding and stability. Subcellular localization analyses and immunoelectron microscopy studies on isolated protein bodies demonstrated that modified γ-zeins accumulate within these organelles and co-localized with endogenous - and γ-zeins. The studies reported here show the feasibility of manipulating the γ-zein gene in order to obtain stable and correctly targeted Lys-rich zeins in maize seeds.

Characterization of the wound-induced metallocarboxypeptidase inhibitor from potato1: cDNA sequence, induction of gene expression, subcellular immunolocalization and potential roles of the C-terminal propeptide

A partial cDNA clone for the potato wound‐inducible metallocarboxypeptidase inhibitor (PCI) was isolated from a cDNA library constructed from mRNA of abscisic acid (ABA)‐treated potato leaves. The full 5′ region of the cDNA was obtained through a RACE‐PCR protocol. PCI mRNA encodes a precursor polypeptide which comprises a 29 residue N‐terminal signal peptide, a 27 residue N‐terminal pro‐region, the 39 residue mature PCI protein, and a 7 residue C‐terminal extension. Northern blot analysis demonstrates that the PCI gene is transcriptionally activated by wounding, and wound signaling can be induced by ABA and jasmonic acid. Subcellular localization of the protein was investigated by immunocytochemistry and electron microscopy, showing that PCI accumulates within the vacuole. A partial PCI precursor form, comprising the mature protein and the C‐terminal extension, has been expressed in Escherichia coli and characterized. Its inability to inhibit carboxypeptidases, and stability to carboxypeptidase digestion, suggest that the C‐terminal pro‐domain may have, besides a probable vacuolar sorting function, a role in modulation of the inhibitory activity of PCI.

Role of structural domains for maize γ-zein retention in Xenopus oocytes

In order to examine the role of cysteine (Cys)-rich domains in the accumulation of maize (Zea mays L.) γ-zein within the endoplasmic-reticulum-derived protein bodies, we studied the localization of γ-zein and of two truncated forms of γ-zein in Xenopus laevis oocytes. The two derivatives were constructed from a DNA encoding the γ-zein: one by deletion of the Pro-X linker region (21 amino acids) and the other by deletion of the Cys-rich domain (94 amino acids). In-vitro-synthesized transcripts were injected into oocytes and the distribution of the translation products was then analyzed. The entire γ-zein and both truncated forms of the γ-zein had accumulated efficiently in microsomes and no traces of secretion were observed. We suggest that neither C-terminal Cys-rich nor Pro-X domains are essential for γ-zein retention in oocyte vesicles. Therefore, structural features derived from disulphide bonds are not necessary for γ-zein targeting on the endoplasmic reticulum.

Molecular characterization of the gene coding for GPRP, a class of proteins rich in glycine and proline interacting with membranes in Arabidopsis thaliana

The gene coding for a new class of proteins rich in glycine and proline (GPRP) was cloned in Arabidopsis thaliana. In the protein sequence, five amino acids-glycine, proline, alanine, tyrosine and histidine-account for 79.4% of the total composition. The protein has two different glycine-rich domains interrupted by a hydrophobic segment having a high probability of helix formation. The protein synthesized in vitro interacts with microsomes possibly through the hydrophobic domain. The gene in Arabidopsis has two introns, one in the coding region and the other one in the 5′ non-coding region. The later one is 778 bp long. Homologous sequences are found in carrot, tomato and tobacco. GPRP mRNA is found in the different organs of the plant analyzed except in mature seeds and anthers, and mostly in epidermal and vascular tissues. Possible hypotheses about the function of GPRP are discussed.

Lysine-rich c-zeins are secreted in transgenic Arabidopsis plants

Abstract. We have previously shown that the maize (Zea mays L.) storage prolamine γ-zein, accumulates in endoplasmic reticulum-derived protein bodies in transgenic plants of Arabidopsis thaliana (L.) ecotype R+P. The retention of γ-zein in the endoplasmic reticulum was found to be mediated by structural features contained in the polypeptide, an N-terminal proline-rich and a C-terminal cysteine-rich domain which were necessary for the correct retention and assembly of γ-zein within protein bodies (M.I. Geli et al., 1994, Plant Cell 6: 1911–1922). In the present work we incorporated in the γ-zein gene lysine-rich coding sequences which were positioned after the N-terminal proline-rich domain and at five amino-acid residues from the C-terminus. The targeting of lysine-rich γ-zeins was analyzed by expression of chimeric genes regulated by the cauliflower mosaic virus (CaMV) 35S promoter in transgenic Arabidopsis plants. The lysine-rich γ-zeins were detected by immunoblotting and we found that these proteins were modified post-translationally to reach their mature form. Subcellular fractionation and immunocytochemical studies demonstrated that glycosylated lysine-rich γ-zeins were secreted to the cell wall of transgenic Arabidopsis leaf cells.

Novel CK2α and CK2β subunits in maize reveal functional diversification in subcellular localization and interaction capacity

In plants, CK2α/β subunits are encoded by multigenic families. They assemble as heterotetrameric holoenzymes or remain as individual subunits and are usually located in distinct cell compartments. Here we revise the number of maize CK2α/β genes, bringing them up to a total of eight (four CK2α catalytic and four CK2β regulatory subunits). We characterize CK2β4, which presents nuclear localization and interacts with CK2α1, CK2α3, CK2β1, and CK2β3. We also describe two CK2α isoforms (CK2α2 and CK2α4) containing N-terminal extensions that correspond to putative cTPs (chloroplast transit peptides). These cTPs are functional and responsible for the subcellular localization of CK2α2 and CK2α4 in chloroplasts. Phylogenetic analysis of the CK2α gene family, further supported by the gene structure and architecture of conserved protein domains, reveals the evolutionary expansion and diversification of this family. The subcellular localization of all four CK2α isoforms was found to be altered when were co-expressed with CK2β, thereby pointing to the latter as regulators of CK2α localization.

Molecular characterization of maize bHLH transcription factor (ZmKS), a new ZmOST1 kinase substrate.

In order to identify potential substrates of the maize kinase in the ABA signalling network, ZmOST1 was used as bait against a library of cDNAs from dehydrated young leaves. A ZmOST1-interactive polypeptide ZmKS (gene locus tag: GRMZM2G114873), showing homology with the Arabidopsis thaliana basic helix-loop-helix (bHLH) DNA-binding transcription factor was identified. Using a comparative genomic approach, the ZmKS corresponding protein was identified as conceptual translated bHLH transcription factor ABA-responsive kinase substrate. ZmKS is localized in the nucleus, shows a potential binding specificity preferentially detectable on cis-acting E-box like heptameric motifs CCACTTG and CAAGTTG, and is phosphorylated by maize protein kinase ZmOST1. ZmKS is expressed in embryo, leaf and root, expression being affected by ABA and osmotic stress. Transgenic Arabidopsis plants, with gain of ZmKS function, show a delay in germination and a transcriptional stomatal opening-facilitator activity, switchover upon ZmKS phosphorylation, suggesting that ZmKS is an ABA-repressed trans-acting activator.

Genes Induced by Abscisic Acid and Water Stress in Maize

The plant hormone abscisic acid (ABA) appears to modulate the responses of plants under conditions of water deficit (Davies and Mansfield, 1983). In general, there are two basic types of response to ABA which have been correlated with regulation of gene expression. First, a slow response during angiosperm embryo development (Quatrano, 1986), with ABA levels increasing (Jones and Brenner, 1987) during the embryogenic period prior to the desiccation of the embryo, and inducing the synthesis of specific proteins and mRNAs (Galau et al. 1987, Sanchez-Martinez et al. 1986). Second, a rapid response in water-stressed plant tissues where the level of ABA increases (Wright and Hiron, 1969) and alters the level of specific gene expression (Heikkila et al. 1984).