Isolation And Functional Characterization Of Rice Stress-Related Transcription Factors

Drought and salinity are major abiotic stresses reducing crop production and qualityand are critical bottlenecks to the development of agriculture in many countries.Therefore, stress resistance has long been targeted in designing better crops. Althoughmany reports have shown that over-expression of some stress-inducible genes intransgenic plants can improve drought and salt resistance, very few are conducted ineconomically improtant crops or tested under the field conditions.The objective of this research is to isolate and characterize some transcriptionfactors conferring drought resistance and to utilize them for genetic improvement ofstress resistance in rice. In this study, large number of stress-inducible transcription factorgenes were over-expressed in rice and transgenic rice were tested for drought resistanceat both seedling and anthesis stages in the field. Based on the evaluation of droughtresistance and functional characterization of a few genes, we found that the SNAC1 andSNAC2 genes showed very significant effect in improving drought resistance and salttolerance in rice. The main results are as follows:1. A total of 33 genes were constructed for over-expression, and 31 genes wereconstructed for RNAi suppression.2. Expression level and copy number of T-DNA were analyzed by northem andSouthern blotting respectively. Results showed that 50% of transgenic plants hadover-expression of transgenes and 30% of transgenic plants had one copy of transgene.3. Compared to the wild type (WT), transgenic seedlings of four target genesshowed drought sensitivity and of five genes showed drought resistance when tested inthe nursery field of rice.4. Transgenic rice of four genes showed increased drought toletance when droughttesting was conducted in the field and PVC pipes at the anthesis stage. According to, theresult of drought testing, we chose SNAC1 and SNAC2 for detail analysis of theirfunctions in drought resistance and/or salt tolerance.5. Northern blot analysis revealed that SNAC1 could be induced in leaves by drought,salt, low temperature, ABA and other stresses. Under drought stress, SNAC1 was predominantly induced in guard cells.6. Transactivation assay, yeast one-hybrid assay, and subcellular localizationanalysis showed that SNAC1 functions as a transcription factor belong to NAC (NAM,ATAF and CUC2) family. The region of amino acid 241-271 is essential to transcriptionalactivation activity. Meanwhile, SNAC1 can bind to the NACRS-like sequence containingCACG and CATGTG core sequences identified in Arabidopsis.7. SNAC1-overexpressing transgenic rice showed increased drought resistance atboth seedling and anthesis stages. Under severe drought stress, seed-setting rate oftransgenic lines could remain more than 20% while that of WT was less than 5%. Nosignificant difference of morphological and agronomic traits was observed betweentransgenic and WT plants.8. SEM analysis indicated that SNAC1-overexpressing transgenic plants have morestomatal closed than WT under normal and drought conditions. Under drought stress,transgenic plants showed delayed leaf-rolling, lost water more slowly, and had lowerminimum relative water content (mRWC) for re-establishing turgor.9. Over-expression of SNAC1 significantly enhanced salt tolerance of transgenic riceat both individual and cellular levels.10. Over-expression of SNAC1 enhanced ABA sensitivity of transgenic plants.11. Many genes related to stomatal movement, osmotic adjustment, cell membranestability and detoxification were up-regulated in the SNAC1-overexpressing transgenicplants.12. T052, encoding a MYB protein, was up-regualted in the SNAC1-overexpressingtransgenic plants. This gene was also induced by abiotic stresses, but the induction levelshowed difference between irrigated rice and upland rice cultivars. Moreover, this genewas expressed in guard cells under drought stress conditions.13. Results of the interaction between SNAC1 and T052 promoter, inducedexpression of both SNAC1 and T052 in guard cells, and increased expression of T052 inSNAC1-overexpressors, all together indicated that T052 was a target gene of SNAC1 mayinvolve in regulation of stomatal closure.14. SNAC2 was induced not only by abiotic stresses (such as drought, high salinity, low temperature and ABA), but also by biotic stress (such as wounding and jasmonicacid). It also functions as a transcription factor belonging to NAC family.15. Over-expression of SNAC2 enhanced salt and cold tolerance and ABA sensitivityof transgenic plants.16. Considering the potential usefulness of SNAC1 in gene engineering for stressresistance improvement, two vectors (pSMDP01 and pSMDP02) for genetictransformation were constructed. SNAC1 was used as a marker gene and OCPI1 promoterwith a bidirectional stress-inducible activity was used to control the expression of boththe marker gene and target gene simultaneously.