| Drought is one of the major abiotic factors limiting plant growth, which induces various changes at the morphologic, physiological, genomic and proteomic levels during adaptation to drought stress in plants. To studying the mechanisms for drought resistance and the adaptation and regulatory mechanisms to drought in plants is crucial for selecting and breeding drought-tolerant cultivars, which could be an important solution to the water shortage and the deterioration of the ecological environment.Drought tolerance of eight bermudagrass genotypes were evaluated in this study, and then two types of turfgrass, which genotypes differing in drought tolerance, warm-season turfgrass'Tifway'(drought-tolerant),'C299'(drought-sensitive), and cool-season turfgrass'Midnight'(drought-tolerant),'Brilliant'(drought-sensitive) were used to investigate photosynthetic response to drought stress. The effects of foliar application of CTKs and KCl on stomatal aperture and photosynthesis after re-watering were examined in Kentucky bluegrass drought-sensitive cultivars. Finally, a subtracted cDNA library was constructed using the approaches of suppression subtractive hybridization (SSH) in leaves of Kentucky bluegrass. A large number of candidate genes which related to drought tolerance were obtained after colony sequencing, BlastX, functional classification and RT-PCR analysis. The main contents and results of this study are as follows:1. Evaluation of drought tolerance in different genotypes of bermudagrassSDS-PAGE analysis demonstrated that no obvious changes in soluble protein profiles occurred in eight bermudagrass genotypes under mild to moderate drought stress. Severe drought stress led to degradation in soluble proteins in eight bermudagrass genotypes, with more significant degradation in'C299'and'Tifeagle'than the other genotypes. No dehydrin polypepides was detected in fully hydrated leaves of well-watered plants. Immunoblot analysis with anti-dehydrin polyclonal antibodies detected one dehydrin polypeptide (24 kDa) in'C299'but not in other cultivars at 6 d of drought. At 10 d of drought stress, six dehydrin polypeptides of 74, 40, 31, 24, 19, and 14 kDa were detected in eight genotypes. The 74-, 19-, and 14-kDa polypeptides accumulated in all genotypes, and no genotypic differences were observed in the level of expression. The 40-kDa polypeptides had lower expression level in'C299','Tifdwarf', and'Tifeagle'. The31-kDa dehydrin polypeptide was detected only in'H19'and'Tifway', but was not expressed in the other six genotypes. The 24-kDa polypeptides expressed in all genotypes, but the intensity in'Tifdwarf','Tifeagle', and'C299'were higher than in the other genotypes. The drought-resistance of eight bermudagrass genotypes were evaluated base on the TQ, RWC, EL, changes of soluble proteins, and the levels of dehydrin expression, the order were as follows: ('Tifway','H19') > ('H10','Kan 1','Sportbermuda','Tifdwarf') > ('Tifeagle','C299').2. Variations in relative water loss rate and expression levels of dehydrin in detached leaves of bermudagrass genotypes differing in drought toleranceDrought-tolerant'Tifway'had a slower RWL than drought-sensitive'C299'during 7 h of dehydration period. The immunoblot analysis indicated that dehydration induced the accumulation of 74-, 40-, 31, 24-, 19-, and 14 kDa dehydrin polypeptides across the RWL range in both genotypes. The intensity of 74-kDa dehydrin polypeptides was did not change with increasing RWL from 10% to 65% in both'Tifway'and'C299'. The intensity of 24-, 19-, and 14-kDa dehydrins increased with RWL from 10% to 65% for both genotypes, but'C299'had higher expression level than'Tifway'at the same water deficit. The accumulation of 31-kDa dehydrin was detected in'Tifway', but not in'C299'during dehydration. As RWL increased from 10% to 65%, the intensity of 31-kDa dehydrin increased in'Tifway'. With the increase in RWL, the intensity of 40-kDa dehydrin proteins was elevated. The accumulation of 40- kDa dehydrin polypeptides in'Tifway'was more pronounced than in'C299'at the same level of water deficit (RWL from 10% to 65%).3. Variations in photosynthetic physiology for two bermudagrass genotypes differing in drought-tolerance under drought stressNo reduction was observed for PSII activity in both'C299'and'Tifway'under mild drought stress (6 d), while significant decrease was observed for Fv/Fm in both genotypes at 10 and 14 d of drought stress, but to a greater extent for'C299'. Drought stress led to a significant decrease in photosynthesis, with more dramatic decline in'C299'than in'Tifway'.'Tifway'maintained significantly higher Pn, gs than'C299'at 6, 10 and 14 d of drought treatment.Rubisco activity in'C299'declined during drought stress to below the well-watered control level at 10 d of treatment, but the decline was not detected until 14 d in'Tifway'. Analysis using SDS-PAGE demonstrated that no obvious changes in Rubisco (49-kDa large subunit) and PPDK (98-kDa) abundance occurred during drought stress compared with the well-watered control (0 d) in'Tifway'. Great change was observed in'C299'for Rubisco and PPDK abundance.4. Effects of drought stress and re-watering on photosynthetic components in Kentucky bluegrassThere was no decline or some reductions in leaf Fv/Fm and other photosynthetic components (Rubisco activity and activation state, Pnmax, CE, Vcmax, Jmax) under short-term or mild drought stress (5 d) in Kentucky bluegrass. Net photosynthetic rate declined with the decreasing of gs, Tr and gm in both'Midnight'and'Brilliant', but to a great extent for 'Brilliant'. Significant decrease was observed in Rubisco activity, activation state, Pnmax, CE, Vcmax, Jmax, in both cultivars under moderate to severe drought stress, with more dramatic decline in'Brilliant'than'Midnight'.Photosynthetic parameters such as Vcmax, Jmax, CE, Pnmax were recovered to or close to the well-watered control level in drought-tolerant'Midnight'after water deficit was alleviated, whereas significantly lower than the well-watered control level for drought-sensitive'Brilliant'. Leaf Pn and gs were still not recovered to the control level after re-watering in both cultivars.5. Effects of CTKs and KCl on stomatal aperture and photosynthesis after re-watering10μM of 6-BA and 50 mM KC1 was sufficient for stomatal re-opening in Kentucky bluegrass after rehydration. Leaf Pn, gs, Tr, and stomatal aperture of Kentucky bluegrass decreased with the decreasing of RWC and SWC under drought stress. Leaf Pn, gs, Tr, and stomatal aperture in 6-BA- or KCl-treated plants significantly higher than untreated plants after re-watering. The decrease in the size of stomatal aperture was highly correlated to the decline in Pn, gs, and Tr under drought stress and during re-watering.6. Isolation of differentially expressed genes in leaves of Kentucky bluegrassA subtracted cDNA library was constructed using SSH approaches, which consist of 503 high quality EST sequences after sequencing. 387 Uni-ESTs were obtained after assembling, which consist of 239 contigs and 148 singlets. BlastX alignment results demonstrated that 156 fragments were homologous with function-known genes, and 147 fragments were homologous with function-unknown genes. The rest 84 ESTs with no hit in published database may represent novel genes. The 156 fragments matched to function-known proteins were further classified into 9 categories, which functions are related to metabolism, protein destination, photosynthesis, transcription, signaling and transduction, cellular defense, cell structure, growth and division, transport, and others. RT-PCR analysis indicated that the expression level of APX, MDHAR, PhGPX increased, whereas the RubS decreased with the progression of drought stress.
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