Identification and differential expression of two thioredoxin h isoforms in germinating seeds from pea.

The NADPH/NADP-thioredoxin (Trx) reductase (NTR)/Trx system (NTS) is a redox system that plays a posttranslational regulatory role by reducing protein targets involved in crucial cellular processes in microorganisms and animals. In plants, the system includes several h type Trx isoforms and has been shown to intervene in reserve mobilization during early seedling growth of cereals. To determine whether NTS was operational during germination of legume seeds and which Trx h isoforms could be implicated, Trx h isoforms expression was monitored in germinating pea (Pisum sativum cv Baccara) seeds, together with the amount of NTR and NADPH. Two new isoforms were identified: Trx h3, similar to the two isoforms already described in pea but not expressed in seeds; and the more divergent isoform, Trx h4. Active recombinant proteins were produced in Escherichia coli and used to raise specific antibodies. The expression of new isoforms was analyzed at both mRNA and protein levels. The lack of correlation between mRNA and protein abundances suggests the occurrence of posttranscriptional regulation. Trx h3 protein amount remained constant in both axes and cotyledons of dry and imbibed seeds but then decreased 2 d after radicle protrusion. In contrast, Trx h4 was only expressed in axes of dry and imbibed seeds but not in germinated seeds or in seedlings, therefore appearing as closely linked to germination. The presence of NTR and NADPH in seeds suggests that NTS could be functional during germination. The possible role of Trx h3 and h4 in this context is discussed.

Differential expression and functional analysis of three calmodulin isoforms in germinating pea (Pisum sativum L.) seeds.

Implication of the ubiquitous, highly conserved, Ca2+ sensor calmodulin (CaM) in pea seed germination has been investigated. Mass spectrometry analysis of purified CaM revealed the coexistence in seeds of three protein isoforms, diverging from each other by single amino acid substitution in the N-terminal alpha-helix. CaM was shown to be encoded by a small multigenic family, and full-length cDNAs of the three isoforms (PsCaM1, 2 and 3) were isolated to allow the design of specific primers in more divergent 5' and 3' untranslated regions. Expression studies, performed by semiquantitative RT-PCR, demonstrated differential expression patterns of the three transcripts during germination. PsCaM1 and 2 were detected at different levels in dry axes and cotyledons, and they accumulated during imbibition and prior to radicle protrusion. In contrast, PsCaM3 appeared only upon radicle protrusion, then gradually increased in both tissues. To characterise the biochemical properties of the CaM isoforms, functional analyses were conducted in vitro using recombinant Strep-tagged proteins (CaM1-ST, CaM2-ST and CaM3-ST) expressed in Escherichia coli. Gel mobility shift assays revealed that CaM1-ST exhibited a stoichiometric binding of a synthetic amphiphilic CaM kinase II peptide while CaM2-ST and CaM3-ST affinities for the same peptide were reduced. Affinity differences were also observed for CaM isoform binding to Trp-3, an idealised helical CaM-binding peptide. However, the three proteins activated in the same way the CaM-dependent pea NAD kinase. Finally, the significance of the single substitutions upon CaM interaction with its targets is discussed in a structural context.