Abstract
Complete submergence is one of the most severe abiotic stresses worldwide, both in natural and man-made ecosystems. A limited number of species have evolved mechanisms to deal with these conditions. Rumex palustris, a model for studying plant responses to flooding, manages to outgrow the water, and thus restore contact with
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the atmosphere, through upward leaf growth (hyponasty) followed by strongly enhanced petiole elongation. These responses are initiated by the gaseous plant hormone ethylene, which accumulates inside plants due to physical entrapment and mimics largely this morphological submergence response. The complexity of ethylene-driven growth in R. palustris upon submergence suggests the involvement of many genes under tight regulation. A comprehensive understanding of the changes in the expression of genes responsive to ethylene is crucial to elucidate the signal transduction networks that lead to hyponastic growth and under-water elongation in this plant. By applying cDNA subtractive hybridisation and cDNA-AFLP (RNA fingerprinting) we identified 119 differentially expressed genes during ethylene-induced hyponastic growth and petiole elongation. From those, 21 have very good homology with Arabidopsis genes. The expression kinetics of these genes were monitored in response to ethylene and submergence using Real Time RTPCR and the putative involvement of candidate genes in (differential) growth during ethylene and submergence treatment was confirmed. Furthermore, we applied whole genome profiling by hybridising mRNA from R. palustris to a CATMA spotted array containing Arabidopsis gene specific tags. This resulted in more than 5000 genes that were differentially regulated in one or both traits. Additionally, the presence of transcription factor binding motifs in the promoters of the differentially expressed genes were examined and demonstrated an over-representation of Abscisic acid responsive elements. Aanalysing the function of putative R. palustris orthologous genes in Arabidopsis during ethylene-induced hyponastic growth revealed that a subset of these genes is likely to control the initial angle of the petiole since, mutations in these genes resulted in constitutive elevated initial petiole angles in Arabidopsis mutant plants compared to the wild type Columbia-0. The others have an effect on either the angle of the petiole or leaf blade angle or both. Mutant analysis revealed a role for PSI-H subunit of photosystem I, glyceraldehyde-3-phosphate dehydrogenase and glutamate synthase in hyponastic growth of Arabidopsis. Furthermore, the effect of auxin response factor ARF8 on hyponastic growth was demonstrated. Reversible protein phosphorylation is a fundamental strategy used by eukaryotes to regulate basic cellular function. Upon a variety of stimuli, kinases phosphorylate downstream kinases usually until the target transcription factor is phosphorylated. Moreover, for many metabolic processes reversible phosphorylation of proteins is a key regulatory mechanism. We examined changes in the kinome profile of R. palustris in response to enhanced levels of ethylene and we discovered differential activity of many kinases. From these experiments, however, we cannot conclude the exact protein kinases involved in ethylene-induced (differential) growth. Our results do show, however, that the kinase regulatory system is strongly regulated during ethylene enriched conditions that induce hyponastic growth and petiole elongation in R. palustris.
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