| For the higher plants, light is not only a source of energy for photosynthesis, but also a key environmental factor that regulates plant growth and development. Plants use phytochromes to perceive and respond to the red (R) and far-red (FR) light which is involved in controlling multiple responses in the plant life cycle from seed germination to fruit ripening. Phytochromes in higher plants are encoded by a small gene family. In rice (Oryza sativa), the phytochrome gene family is composed of three members, PHYA, PHYB, and PHYC. The characterization of rice phytochrome mutants has revealed that phyB mutants (phyB) exhibited improved drought tolerance. Therefore, the phyB mutants can be regarded as an important germplasm resource with improved drought tolerance.In this study, to explore underlying mechanism by which phyB regulate the drought tolerance, the physiological characters such as water uptake and water loss of the roots and shoot of the phyB mutants and wild type and genes related to drought tolerance were examined. The main results were as follows.1. phyB mutants of rice exhibited improved drought toleranceMost of WT plants were wilting, while the phyB mutants were not after the phyB1 mutant and WT plants at the six-leaf stage were water-withheld for 13 days. After being water-withheld for 16 days and then being re-watered for 12 days, 90% of phyB mutant plants regrew and produced new leaves, while only 47.5% for the WT plants. These results clearly demonstrated improved drought tolerance in the phyB-deficient mutants. We measured the proline contents and soluble sugar content in wild type and phyB mutants grown either under the normal condition or 15% PEG4000-treated condition. It was shown that both proline and sugar contents were higher in phyB mutants than in wild type under both the normal and PEG-treated condition. Therefore, it indicated that the phyB mutants have higher osmotic adjustment capacity than wild type to cope with drought stress.2. phyB mutants and WT have similar root charecteristicsThe length of seminal roots and ratios of root to shoot in WT and phyB mutant seedlings grown in 0.5% agar medium at the two-leaf stage were measured. It was revealed that phyB mutants exhibited longer seminal roots than WT, whereas ratios of root to shoot were not significantly different. We further analyzed the root system in WT and phyB mutants grown in nutrient soil at the six-leaf stage. Root lengths of phyB mutants were not statistically different from that of wild type. Investagation on root system of phyB mutants and wild type at the six-leaf stage by root system analyzer (WinRHIZO) revealved that root length, suface root area and root diameter were similar in the WT and phyB mutant. Therefore, it is believed that growth and uptake surface area of roots were probably not a major factor contributing to drought tolerance in the phyB mutants.3. phyB mutants exhibited reduced water loss rate and transpiration rateThe fifth detached leaves of the 3 phyB mutants (phyB1,phyB2,phyB5) and WT plants at the six-leaf stage were used to compare water loss rate. Leaves of phyB plants showed an obviously slower rate of water loss than WT leaves. Net photosynthesis rate, stomatal conductance and transpiration rates of WT and the phyB mutants were futher determined using potable phtosynthesis analyzer (LI-6400), and the results showed that both photosynthesis rate and the transpiration rate were lower in the phyB1 mutant as compared to those in WT, which is consistent with the phenotypes of stomatal density and length. However, WUE of the phyB mutant and WT was not significantly different. It suggseted that reduced water loss from leaves is likely one of the major factors contributing to drought tolerance in the phyB mutants. In addition, the total leaf area of phyB mutants was obviously smaller than that of wild type. These results suggest that phyB mutants have the lower transpiration rate than WT under the same growth condition, which is possibly the key factor for phyB mutants have higher drought-tolerance.4. phyB positively regulates stomatal densityThe number and size of stomata in the third, fourth, and fifth leaves of WT and the phyB1 mutant at the six-leaf stage were measured. Stomatal density of phyB mutant was significantly lower and stomatal length was markedly smaller that those of WT based on the imprint images of adaxial epidermis. These results suggest that the deficiency of phyB causes a decreased stomatal density and size, which will reduce photosynthesis, transpiration and water loss rate.5. phyB down-regulate the expression of putative ER family genes controling stomata developmentTo explore the molecular mechanisms of the reduced stomatal density and stomatal size in the phyB mutants, we examined the expression profiles of genes related to the stomatal development in primordial leaves and mature leaves of WT and phyB mutants at the stage of 2nd and 4th day of seed germination and 6th leaf by real -time PCR. Results showed that three genes (ER,ER1/2 and ERL) homologous to members of the Arabidopsis ER family always expressed higher in phyB mutants than in wild type, suggesting that phyB down-regulates the expression of putative ER family genes in rice. ER family was reported to be negative regulators of stomatal development in Arabidopsis. Therefore, we hyphothesize that phyB regulates the stomatal density and size by regulating the expression of ER family gene in rice.6. ERECTA negatively regulate the transpiration in transgenic tobaccoTo further access the role of rice ER family gene on the transpiration rate, plant transformation vector pCAMBIA1390-Ubi-OsERL was constructed and transferred into tobacco. After water was withheld for 18 days, several independent transgenic lines were shown to keep turgor while wild type was obviously wilt, and water loss of the detached loeaves of transgenic lines was significantlt lower thant that of wild type. Net photosynthesis rate, stomatal conductance and transpiration rates of WT and the phyB mutants were futher determined using potable phtosynthesis analyzer (LI-6400), and the results showed that transgenic lines exhibited lower transpiration rate and higher and water use effiency (WUE) than wild type while photosynthesis rate was not significantly different. These results suggest that overexpression of OsERL confers drought tolerance possibly via reducing transpiration rate and water loss rate. After analyzing the stomata number and epidermal cell size on the 6th expanded leaves, it was observed that transgenic lines have lower stomatal density and lager epidermal cell compared towild types, but stomatal morphogenesis was similar. These results suggest that ERECTA increased epidermal cell size and reduced stomatal density to reduce the transpiration rate.7. phyB–mediated red light signal transduction represses expression of ERECTA genesTo analyze how phyB regulates the expression of ERECTA genes, the expression levels of three ER family genes in wild type and phyB mutants grown either under dark condition or under continous red light were dermined. For the wild type, expressions of ER genes were obviously repressed by red light. However, the repression effect triggered by red light was weakened in phyB mutants. These results suggested that phyB–mediated red light signal transduction represses the expression of ER family genes in rice.8. ABA-dependent pathway is involved in the improved drought tolerance of phyB mutantsABA is an important stress hormone. To investigate the involvement of ABA in phyB-regulated drought responses, we compared the sensitivity of wild type and phyB mutant to exogenous ABA based on the seed germination assay. It was revealed that phyB mutant is hypersensitive to ABA. The expression of ABA metabolic genes in wild type and phyB mutants were analyzed using real-time PCR. ABA biosynthesis genes (ZEP and NCED) expressed higher in the phyB mutant than in wild type, whereas the ABA degradation genes expressed lower in phyB mutant than in wild type. These results suggest that phyB down-regulates the expression of ABA biosynthesis genes and up-regulates the expression of ABA degradation genes.The ABA metabolic gene profiles of wild type and phyB mutants of drought for different times were compared, it was found that expression levels of several genes involved in ABA signal pathway were significantly higher phyB mutants than in wild type, such as osmotin, PP2C family gene, bZIP transcriptional factor genes and stress-induced genes. These results suggest that ABA-dependent pathway is probably involved in the regulation of improved drought tolerance of phyB mutant.In summary, phyB-mediated mechanism of drought-tolerance has been invesrigated at different levels of the physiological characters such as root growth, water loss and genes related to stomata development and ABA metabolism. It concluded that on one hand phyB down-regulates ERECTA expression leading to increase of epidermal cell size, decrease of stomata density and transpiration. On the other hand, phyB up-regulates ABA-synthesis gene expression and down regulates ABA-catabilism gene expression leading to improvement of drought tolerance via ABA-dependent pathway. Taken together, we hypothesize that improved drought tolerance in phyB mutants is, on one hand, attributed to the up-regulated expression of the negative regulators (ER family genes) involved in stomata development. On the other hand, phyB also affects ABA-dependent pthway via regulating the expression gene related to ABA metabolism and ABA-dependent pathway. |
