As a usual response, plants induce/activate various proteins which are thought to be involved in defense mechanisms against the biotic and abiotic stresses they may be confronted with. The novel DUF538 domain containing proteins with unknown functions have been found to be induced/activated in response to different environmental stress stimuli in plants. In order to perform biochemical studies with these new plant stress-responsive proteins, a cDNA containing DUF538 domain was amplified from Celosia cristata full-length leaf expression library using a specific primer set. The isolated cDNA was subsequently expressed in Escherichia coli as a part of maltose-binding fusion protein (MBP-DUF538 construct) and purified at the yield of about 32 mg per liter of cell culture by affinity chromatography without affecting the recombinant bacterial cell growth. The purified fusion product was exogenously applied (10 μg per 4 cm(2)) on the leaves of Nicotiana tobaccum L. The results revealed that fused DUF538 protein does not induce morphological reposes, but elevates redox enzyme activities including catalase, peroxidase, polyphenol oxidase and phenyalanine ammonia lyase. This is the first time ever time report with respect to the heterologous expression of a plant stress-responsive DUF538 domain that may provide a basis to study its physiological roles and biochemical activities in vitro and in vivo.

Differential antioxidative activities were assessed in the leaves of two maize inbreds (A-180 and A-619) under salt stress and the subsequent recovery period. Total antioxidation test revealed that in both inbreds, this ability was sharply increased during stress period, but was slowly reverted back to the normal level during recovery. The enzymatic antioxidative analysis showed differential patterns in the activities of catalase, peroxidase and polyphenol oxidase in both maize inbreeds. Comparative analysis of the activity of phenylalanine ammonia lyase (PAL), a key enzyme at the gateway of propanoid biosynthetic pathway, suggested that propanoid compounds might be antioxidants of pivotal importance to the salt-challenged maize antioxidation system. As for drought-stressed plants, a PAL-dependent antioxidative strategy is proposed as a promising target for maize salt resistance engineering.

The possible hydrolytic activity towards chlorophyll molecules was predicted for DUF538 protein superfamily in plants. It was examined by using computational as well as experimental tools including in vitro chlorophyll degradation, antioxidant compounds production and in vivo real-time gene expression tests.Comparison of the computational data with the experimental results indicated that DUF538 proteins might be chlorophyll hydrolyzing enzyme (most probably carboxyesterase) which degrade chlorophyll molecules (66 % per 12 hrs) to produce new compounds (1.8 fold per 12 hrs) with antioxidant properties. The relevance of DUF538 gene expression level with the chlorophyll contents (2.8 fold increase per chlorophyll content of 50 %) of the drought-stressed leaves showed that chlorophyll degradation by DUF538 is most probably induced in response to stress stimuli. Despite membranous chlorophyll catabolic pathways, DUF538-dependent reactions is predicted to be occurred in the cytosol of the under stressed plants. We addressed as to whether chlorophyll breakdown to antioxidant compounds by DUF538 is a defense mechanism of plants against stress stimuli, in vivo? This question is going to be investigated in our next research project.

DUF538 protein super family includes a number of plant proteins that their role is not yet clear. These proteins have been frequently reported to be expressed in plants under various stressful stimuli such as bacteria and elicitors. In order to further understand about this protein family we utilized bioinformatics tools to analyze its structure in details. As a result, plants DUF538 was predicted to be the partial structural homologue of BPI (bactericidal/permeability increasing) proteins in mammalian innate immune system that provides the first line of defense against different pathogens including bacteria, fungi, viruses and parasites. Moreover, on the base of the experimental data, it was identified that exogenously applied purified fused product of Celosia DUF538 affects the bacterial growth more possibly similar to BPI through the binding to the bacterial membranes. In conclusion, as the first ever time report, we nominated DUF538 protein family as the potential structural and functional homologue of BPI protein in plants, providing a basis to study the novel functions of this protein family in the biological systems in the future.

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