Diagonal two-dimensional electrophoresis (D-2DE): a new approach to study the effect of osmotic stress induced by polyethylene glycol in durum wheat (Triticum durum Desf.).

Acclimatization to stress is associated with profound changes in proteome composition. The use of plant cell and tissue culture offers a means to investigate the physiological and biochemical processes involved in the adaptation to osmotic stress. We employed a new proteomic approach to further understand the response of calli to dehydration induced by polyethylene glycol (PEG6000). Calli of three durum wheat genotypes Djenah Khetifa, Oued Zenati and Waha were treated with two concentrations of polyethylene glycol to mimic osmotic stress. Changes in protein relative abundance were analyzed using a new electrophoretic approach named diagonal two-dimensional electrophoresis (D-2DE), combined with mass spectrometry. Total proteins were extracted from 30-day-old calli from three durum wheat genotypes that showed contrasting levels of drought stress tolerance in the field. The combination of one-dimensional electrophoresis and D-2DE gave a specific imprint of the protein extracts under osmotic stress, as well as characterizing and identifying individual target proteins. Of the variously expressed proteins, three were selected (globulin, GAPDH and peroxidase) and further analyzed using qRT-PCR at the transcriptome level in order to compare the results with the proteomic data. Western blot analysis was used to further validate the differences in relative abundance pattern. The proteins identified through this technique provide new insights as to how calli respond to osmotic stress. Our method of study provides an original and relevant approach of analyzing the osmotic-responsive mechanisms at the cellular level of durum wheat with agronomic perspectives.

Apple messenger RNAs related to bacterial lignostilbene dioxygenase and plant SAUR genes are preferentially expressed in flowers.

In an attempt to use a differential display procedure to identify organ-specific genes in apple, cDNA fragments of two transcripts preferentially expressed in flowers were isolated and corresponding full-length cDNA inserts were subsequently obtained. One of these clones, Md-FS1, belongs to the SAUR gene family, originally identified as a set of auxin-inducible genes in soybean. The second one, Md-FS2, encodes a polypeptide with sequence similarities to bacterial lignostilbene-alpha,beta-dioxygenase isozymes, which are thought to be involved in lignin biodegradation. Northern blot analysis confirmed that both genes are preferentially expressed in floral organs at full bloom, while being expressed at lower or undetectable levels in vegetative organs (leaves, shoots or roots) as well as in immature, green and unopened blossoms. Furthermore, Md-FS1 transcripts also appeared to accumulate in vegetative tissues after auxin treatment of micropropagated apple shoots.

Knotted1-like homeobox genes are expressed during apple tree (Malus domestica [L.] Borkh) growth and development.

Apple (Malus domestica [L.] Borkh) cDNA clones encoding two distinct but very similar kn1-like homeobox gene class 1 homologues (KNAP1 and KNAP2) were isolated using a fragment amplified with degenerated primers as a probe. A fragment corresponding to the homeodomain region of KNAP1 was used to isolate a cDNA (KNAP3) belonging to the kn1-like homeobox gene class 2. These probes were used to detect corresponding gene copies in apple genomic DNA digests, together with other putative members of an apple kn1-like homeobox gene family. The kn1-like homeobox gene probes were also used to detect corresponding messenger accumulation levels in various organs of an apple tree. Transcripts corresponding to KNAP1 and KNAP2 genes appeared to be absent from leaves or floral organs, but they accumulate in tissue samples from elongated parts of the stem. In contrast, KNAP3 mRNAs accumulate at detectable levels in a wider range of both vegetative and reproductive organs.

Developmental and circadian pattern of rubisco activase mRNA accumulation in apple plants.

An apple cDNA encoding the precursor of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase has been characterized. Using this cDNA as a probe, leaf-specific and light-regulated accumulation of corresponding transcripts was detected. Rubisco activase transcripts also turned out to accumulate at growing levels during apple leaf development, to reach a maximum in fully expanded leaves. In contrast, chlorophyll a/b-binding protein (Cab) and rubisco large subunit mRNA levels reach a maximum earlier in the course of leaf development. Moreover, the accumulation of rubisco activase messengers appeared to follow an oscillating circadian rhythm qualitatively similar to that observed for Cab mRNA levels.

A calcium/calmodulin-binding serine/threonine protein kinase homologous to the mammalian type II calcium/calmodulin-dependent protein kinase is expressed in plant cells.

cDNA fragments corresponding to an apple (Malus domestica [L.] Borkh) calmodulin-binding polypeptide have been isolated and characterized. The protein encoded by this messenger contains a serine/threonine protein kinase catalytic domain followed by a calcium/calmodulin-binding regulatory domain, both exhibiting significant sequence similarities to the corresponding regions of the mammalian calcium/calmodulin-dependent protein kinase II subunits. These results confirm a potential regulatory role for calmodulin in phosphorylation-mediated signal transduction events.

Characterization of Two Gene Transcripts Modulated by Cytokinins in Micropropagated Apple (Malus domestica [L.] Borkh) Plantlets.

The micropropagation of apple (Malus domestica [L.] Borkh) cultivars is usually achieved by axillary bud stimulation and requires an exogenous cytokinin supply. Two cDNA libraries were constructed from mRNA isolated from plantlets grown in vitro on medium with or without benzyladenine. One cDNA clone (pSD3), corresponding to transcripts more abundant in plantlets grown on medium containing cytokinin than on medium lacking the hormone, was isolated. It corresponds to a mRNA of about 1,800 nucleotides which codes for a proline-rich protein with a predicted mass of 31,000 daltons. Its accumulation is restricted to roots and stems of in vivo grown apple plantlets and to stems of microcuttings cultivated on medium without cytokinin. Furthermore, it accumulates to comparable levels in leaves and stems when plantlets are grown on medium containing benzyladenine. A second cDNA clone (pSD4), corresponding to transcripts down-regulated in the presence of cytokinin in the culture medium, was also characterized. Its corresponding mRNA is about 700 nucleotides in length and encodes a protein highly homologous to the precursor of the 10-kilodalton polypeptide of the photosystem II from spinach. This mRNA accumulates specifically in leaves of apple plantlets and is more abundant in leaves of plantlets grown in the absence of cytokinin compared with plantlets grown in the presence of benzyladenine.