STRESS RESPONSES AND ADAPTAION MECHANISMS IN PLANTS: REGULATION OF CELL DIVISION CYCLE AND PROTECTIVE PATHWAYS

Under extreme environmental conditions the reduction in growth of plant organs and productivity is highly dependent on the functionality of basic cellular mechanisms including cell division or defence reactions. The primary aims of ongoing research projects are the identification and characterization of genes, protein complexes involved in the control of cell cycle progression and sensing hormonal and stress signals. Comparison of stress sensitive and resistant genotypes from wheat and barley is expected to reveal novel mechanisms in adaptation to oxidative stresses or drought. Brachypodium distachion, a new model for temperate grasses is also used as experimental system in studies on linkage between drought adaptation and root development. In our candidate gene approach much attention is devoted to the analysis of the stress-protective function of rice and wheat aldo-keto reductases.

Plant retinoblastoma-related proteins as key regulators of cell division and growth

Cell division is one of the most fundamental cellular events that contributes to the realization of the developmental program and organization of plant structure. In plants as in other eukaryotic organizations, the cell division cycle includes the replication of DNA molecules in S-phase and subsequent organization of chromosomes, which are equally distributed to the daughter cells during mitosis (M-phase). Two major checkpoints, the first before entering S-phase at the end of G1-phase and the second before mitosis at the end of G2-phase control the progression through the cycle. Figure 1 proposes a central role for the retinoblastoma-related proteins (RBRMs) in the regulation of the G1-S transition through interaction with cyclin-cyclin-dependent kinase (CDK) and phosphatase (PP2A) complexes. The regulatory function of RBR depends on its phosphorylation status and interaction with E2F/DP transcriptional factors. Based on our research data the described model suggests alternative ways for the stress and hormone-activated Ca2+ signals in blocking G1-S transition in alfalfa (Medicago sativa, Ms) cells. The Ca2+-activated PP2A phosphatase complex can ensure RBR dephosphorylation resulting in the block in the binding of the E2F/DP transcriptional factors, consequently the induction of S-phase genes is inhibited. We also showed that CDK complexes can phosphorylate RBR protein from alfalfa. This modification is expected to release the E2F/DP factors for activation of S-phase genes. The CDK function is under the control of the inhibitor protein (CKIMs) that is also a phosphoprotein and substrate of the Ca2+-dependent protein kinase (CDPKMs). The cellular Ca2+ level can be increased under hormonal or stress effects. We showed that the stress hormone abscisic acid (ABA) reduced CDK activity in cultured alfalfa cells. In contrast, auxin and cytokinins activate this kinase primarily responsible for RBR phosphorylation and activation of genes in S-phase

Cloning of the rice RBR cDNAs (OsRBR1 and OsRBR2) allowed the production of transgenic cells or plants with overproduction or down-regulation in the synthesis of the corresponding mRNAs (Figure 2). The reduced synthesis of OsRBR1 protein caused significant increase in the frequency of S-phase cells, whereas overproduction decreased the number of DNA synthesizing cells, as shown by Figure 2. Cultured cells of antisense line exhibited higher cell mass production in suspension culture. We consider retinoblastoma-related proteins to be central regulators of plant cell division cycle and our present research is focused on the role of phosphorylation, degradation of RBRs in the control of plant growth, development and stress adaptation.

Drought responses in cereals: a genomic approach

Plants have developed a wide range of defence strategies to maintain the functional integrity of cells and the whole organism under drought stress. Efficient root system, effective ROS eliminating pathways both play important role in surviving periods of water shortage. We aim to reveal key molecular components of long term drought adaptation of the root system in cereals. Remote wheat genotypes having different root growth characteristics (Figure 3) and upland rice cultivars are studied in genome wide transcript profiling experiments.

Comparative transcriptome analysis of a spring variety, Kobomugi with a winter variety Plainsman that follows avoidance strategy revealed that stress and defence related genes are more frequently up-regulated in the first one, while cell wall, growth and signal transduction-related transcripts are more abundant in the second one.

Transcriptome analysis of upland rice cultivars highlighted genes exhibiting daily fluctuation in induction during drought. A LEA type gene is not only induced proportionally to the extent of stress, but strictly root specific as well. Its promoter was fused to reporter gene construct and transformed into cereals to evaluate practical usability. Groups of co-ordinately regulated rice genes allow us to search for common regulatory elements in the whole promoterome using our computer algorithm that finds new common elements in drought induced genes.

ROS eliminating machinery of wheat plays important role in aerial part of plant as well. Comparing responses of two wheat genotypes, we found differences in ascorbate metabolism: both ascorbate oxidation and transcription levels of enzymes processing ascorbate were changed. Relative transcript levels of ascorbate peroxidase, mono dehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase isoenzymes showed different transcriptional changes in the two genotypes.

Brachypodium distachion, a new model for temperate grasses is also used to implement our experimental system and we will study linkage of drought adaptation and root development.

Reactive carbonyl detoxification and stress tolerance in plants

The productivity of plants is greatly affected by environmental stresses such as drought, increased soil salinity, high or low temperature. The production of reactive oxygen species (ROS) and their toxic products, the reactive carbonyl compounds (RCC) are common feature of most environmental stresses, their reaction with proteins can significantly inhibit the normal activity of basic enzymes such as phosphoribulo kinase (Figure 4/A).

Improvement of scavenging capacity of cells can lead to increased stress tolerance. Plant aldose/aldehyde reductases are important factors for such function since they have a wide range of enzymatic activity on lipid peroxidation and glycolysis derived reactive carbonyls (like 4-hydroxy non-2-enal and methylglyoxal) and their detoxification capacity can limit the harmful effects of such stress-derived compounds. Our main goals are: i, analysis of the molecular background of enzyme modifications by reactive carbonyl compounds; ii, searching for candidate genes that can be used in the detoxification process; iii, based on the results improve stress tolerance of important crop plants. According to our previous results transgenic tobacco plants ectopically expressing the alfalfa aldose reductase, the MsALR cDNA under the control of the CaMV 35S promoter were more tolerant to dehydration stress and recovered better from damages caused by water deficit than the untransformed wild type plants and were more tolerant to stress caused by high temperature and light intensity or UV-B irradiation. Analysis of transgenic wheat plants over-producing the alfalfa enzyme by the complex diagnostic system developed in collaboration with the Cereal Research Non-Profit Co. revealed significant improvements in agronomical parameters during drought stress (Figure 4/B). This result supported the start of a conventional breeding program that aims the improvement of stress tolerance of Hungarian wheat varieties.