3 Results

3.1 Plant responses to drought stress

At 157 day after sowing, the shoot dry weight and leaf relative watercontents were markedly
decreased by water deficit in both genotypes. In drought stressed plants, shoot dry weight was
approximately 50% of the irrigated controls in both genotypes. The leaf relative water content in
stressed plants was decreased to 65% in both genotypes. Thus, in this study the two genotypes showed
similar responses to drought and both experienced similar levels of water deficit. However, proteomic
analysis may show subtle differences in drought stress responses that are not evident from
measurements of dry matter and leaf water status.

3.2 Analysis of proteins altered under stress

From more than 500 spots were reproducibly detected on silverstained gels, 77 spots showed
significant changes in both stressed and control leaves in genotype 7112 In addition, two proteins (78,
79) were visible only in CBB stained preparative gels in stressed plants. Considering both genotypes,
there were 79 responsive proteins: 27 were upregulated, 44 were downregulated, and eight proteins
were detected only in stressed plants. We classified the 71 responsive proteins that were detected in
both treatments into several groups. The major group was composed of 26 proteins that were
downregulated under stress in both genotypes. Twelve proteins were downregulated only in 7219-
P.69, whereas seven proteins were downregulated only in 7112. Ten proteins were upregulated in
both genotypes, whereas six and seven proteins were upregulated only in 7219-P.69 and 7112,
respectively. Three spots, which were detected only in genotype 7112, were upregulated under stress.
In summary, the number of downregulated proteins and the degree of down-regulation was higher in
genotype 7219-P.69.

Protein identification

Twenty drought-responsive proteins were examined by LCMS/ MS. Spots that showed consistent
positions on different gels were considered to be the same proteins. Standard marker proteins also
were found at the same positions on different gels. These included two proteins detected only under
water deficit conditions (heat shock protein (HSP) and putative oxidoreductase), and eight upregulated
proteins (2-cysteine peroxiredoxin (2-Cys Prx), superoxide dismutase (SOD), nucleoside-diphosphate
kinase (NDPK), hypothetical protein, and four Rubisco fragments), and one downregulated protein
(putative nascent polypeptide-associated complex a-chain (a-NAC)). Where Mascot failed to provide
identification, spectra were interpreted both manually and using sequencing algorithms. The derived
amino acids sequences were subject to EMBL MS BLAST homology- based searching leading to
identification of four additional proteins that were detected only in drought stressed plants: class II
heat shock protein, osmotin-like protein, and two cyclophilins

Comparison with drought-related proteins of maize, watermelon, and rice

We identified four rubisco fragments and 11 other proteins. Eleven other proteins identified in this
study diverged considerably from drought responsive proteins identified in maize (16 proteins), rice
(16 proteins), and wild watermelon (two proteins) [1517]. This divergence may be due to the fact that
only a small portion of drought responsive proteins were identified in sugar beet (15 out of 79), maize
(16 out of 78), and rice (16 out of 42). The identification of other responsive proteins would provide us
with a better understanding of common and different drought responsive pathways. This divergence
may also reflect the fact that the experiments were conducted under different conditions with
different genetic materials. The results of proteome analysis of maize, rice, and sugar beet revealed
substantial genetic variation in the expression of the drought response even within species.