Candidate proteins retrieved in the Oregon Wolfe Barley population

Barley accessions from the OWB mapping population exhibit a contrasting response towards salinity stress during germination. In a comparative proteome analysis of mature grains from these genotypes 6 protein spots were identified differing in abundance between salt tolerant and salt sensitive genotypes.

In germination assays it could be shown that the parent lines of the OWB population, DOM and REC, display a contrasting response for salinity at the germination stage (Weidner et al., 2005). Therefore, mature grains were chosen for a comparative proteome approach aiming at the identification of proteins conferring salt tolerance during germination. A comparison of the protein patterns of the parent lines DOM and REC revealed a high number of spots differentially expressed in the grain. To limit the number of candidate proteins and to take advantage of the huge genetic resource that mapping populations represent, 4 additional lines were added to the comparison showing an even stronger trait for salt response as the parent lines. In the comparative proteome analysis including all 6 lines, only 11 spots showed differential expression between groups of salt tolerant and salt sensitive lines. The reason for this reduction is the large heterogeneity of the spot patterns due to genotype-specific protein spots. By excluding these spots, presumably contributing to morphological or other characteristics, and by comparing only spots that were common to all lines, the number of candidate spots was limited to a feasible size with the protein identification successful for 6 spots (Table 8).

Table 8: Summary of identified proteins from the comparative analysis of mature grains from accessions of the OWB population showing contrasting response towards salinity at the germination.

Spot

number Protein name Expression in salt tolerant genoytpes 3 Cytosolic 6-phosphogluconate dehydrogenase ↑

6 Glucose and ribitol dehydrogenase homolog ↑ 7 Putative elongation factor β ↓

8 Hsp 70 ↓

9 Glucose and ribitol dehydrogenase homolog ↑ 10 Translationally-controlled tumor protein homolog ↓

Among these identified protein spots, spot # 3 showed a higher abundance in salt tolerant lines and was identified as cytosolic 6-phosphogluconate dehydrogenase (6PGDH). This enzyme plays a key role in the pentose phosphate pathway, which provides reduced nicotinamide adenine dinucleotide phosphate (NADPH) for metabolic and detoxification reactions (Figure 56).

Figure 56: Schematic diagram of the oxidative pentose phosphate pathway. While CO2 fixation takes place in the reductive pentose phosphate pathway, the oxidative pentose phosphate pathway yields in the oxidation of a hexosephosphate to a pentosephosphate under the release of CO2. The significance of this pathway is the production of NADPH.

fructose-6-phosphate

glucose-6-phosphate

6-phosphogluconolactone CO₂fixation (Calvin cycle)

NADP⁺

NADPH+H⁺ glucose-6-phosphate dehydrogenase

6-phosphogluconate

ribulose-5-phosphate

NADP⁺

NADPH+H⁺ 6-phosphogluconate dehydrogenase 6-phosphogluconolactonase

H₂O H⁺

phosphoglucose isomerase

CO₂

NADPH is a reducing equivalent necessary as electron donor in reductive biosynthetic reactions like synthesis of lipids, aromatic amino acids and coenzymes. It is also essential in the ascorbate-glutathione cycle, which is an important antioxidant protection system (Corpas et al., 1998). Recent studies confirmed the relation between oxidative stress upon salt treatment and enhanced expression of NADPH-dependent dehydrogenases. Olive plants were subjected to salt stress and consequently, the protein content and activity of glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase and malic enzyme, all NADPH-recycling enzymes, increased significantly (Valderrama et al., 2006). In shoots of salt-treated rice plants, transcripts of 6PGDH were up regulated under stress conditions as early as 30 min after treatment (Huang et al., 2003). The accumulation of 6PGDH in grains of salt-tolerant barley lines could therefore indicate an enhanced pentose phosphate pathway and a better supply with NADPH leading to an accelerated germination under stress conditions.

Furthermore, the protein could function as protection against oxidative damage caused by salinity.

Two spots (# 6 and 9) were identified as glucose and ribitol dehydrogenase homolog (GlucDH) that were more abundant in salt tolerant lines. The sequence of the identified protein shared similarities with glucose dehydrogenase and ribitol dehydrogenase from bacteria, which are structurally related and belong to the short alcohol dehydrogenases (Jornvall et al., 1984). Transcripts and protein of GlucDH were found exclusively in developing barley embryos and levels decreased during germination (Alexander et al., 1994).

Enzymatic measurements revealed the restriction of dehydrogenase activity to the oxidation of D-glucose, but not sugar-phosphates, using NAD as co-substrate (Alexander et al., 1994).

This points to a metabolic pathway not yet described in barley and could indicate a specific carbohydrate metabolism important for early embryo development. The higher abundance of GlucDH in grains of salt tolerant genotypes points to an enhanced carbohydrate metabolism.

The identification of GlucDH in two distinct protein spots with different pI and molecular weight could result from posttranslational modifications or an additional isoform. In fact, the position of spot # 6 corresponds to the theoretical biochemical properties deduced from the amino acid sequence, whereas spot # 9 shows a lower molecular weight and a higher isoelectric point. Peptide mass fingerprinting and de novo sequencing did not reveal further insights into the structural integrity of the protein and it remains open whether a protein modification or an enzyme isoform is responsible for the multiple observation of this protein.

Spot # 7 was less abundant in salt tolerant lines and identification revealed a putative elongation factor 1β (EF-1β). This protein appeared as low temperature responding in cold-treated barley leaves (Baldi et al., 2001). Components of the protein translational apparatus, including EF-1β, showed an accumulation at the transcript level in frost sensitive and resistant plants and authors concluded that the up regulation of translation machinery for synthesis of soluble proteins during cold acclimation is a general response reaction. The findings from the comparative proteome approach could indicate an enhanced synthesis of stress-related proteins in the sensitive accessions.

A translationally controlled tumor protein homolog (TCTP, spot # 10) was more abundant in salt-sensitive lines. TCTP is described as a calcium-binding protein abundant in various animal tumor cells and homologous proteins have been found in a wide range of organisms including human, mouse, yeast and plants. The expression of TCTP in Pharbitis was related to photoperiodism (Sage-Ono et al., 1998) and in soybean, transcripts accumulated after aluminum-induced stress (Ermolayev et al., 2003). The high level of conservation indicates an essential function, but at this stage it is difficult to suggest a possible function during salt-stress in susceptible plants.

Spot # 8 was exclusively found in salt-sensitive barley lines and identified as Hsp 70, a molecular chaperone that is highly conserved in plants. In Arabidopsis, at least 18 genes encoding members of a 70 kDa heat shock protein family are known with developmental and environmental regulated expression (Lin et al., 2001). Besides the known induction by heat and cold stresses, it plays also an important role in grain development and germination (Sung et al., 2001). During grain desiccation, proteins are unfolded or misfolded and Hsp 70 might be necessary to ensure proper protein aggregation in the ripening grain. The accumulation of Hsp 70 in the salt sensitive line is in contrast to the previous findings, but it could confer tolerance to other stress factors, like drought, which has not been tested yet.

The comparative proteome analysis with accessions from the OWB population was performed to identify proteins that might confer salt tolerance during germination. Two proteins were found higher expressed in salt tolerant lines as compared to salt sensitive accessions. These were 6PGDH and GlucDH and both were selected for stable transformation experiments to test the effect of constitutive and grain-specific overexpression in a salt sensitive barley cultivar (Golden Promise). Northern blot analysis of non-transgenic plants revealed a transcript accumulation of GlucDH in mature grains, but not in leaf tissue, whereas 6PGDH transcripts were detected in grains and leaves to a similar extent. This indicates the

grain-specificity for GlucDH, but not for 6PGDH. After screening the T0 generation of transgenic plants on the genome and transcriptome level, transgenic lines were selected for propagation.

The T1 and T2 progeny will be used in prospective experiments to test the transgenic plants for their enhanced ability to germinate during salt stress application.