Down-regulation of GhADF1 in cotton(Gossypium hirsutum)improves plant drought tolerance and increases fiber yield

Fiber productivity and quality of cotton are severely affected by abiotic stresses.In this study,we identified the role of GhADF1,an actin depolymerizing factor,in cotton response to drought stress.GhADF1 expression in cotton could be induced by PEG6000.GhADF1-RNAi transgenic cotton showed increased tolerance to drought stress during seed germination and seedling development as well as at the reproductive stage.In contrast,overexpression of GhADF1 led to a drought-sensitive phenotype in transgenic plants.GhADF1-RNAi plants produced an enlarged root system with longer primary roots,more lateral roots,increased root dry biomass,and increased cell size.In leaves of GhADF1-RNAi cotton,proline content and activities of reactive oxygen species-scavenging enzymes were increased following drought stress compared with those in wild type.GhADF1-RNAi lines showed higher water-use efficiency than the wild type,accompanied by reduced leaf stomatal density and conductance.GhADF1-RNAi cotton produced higher fiber yield in the field under both normal and drought conditions.Transcriptomic analyses identified 124 differentially expressed genes in leaves of GhADF1-RNAi lines compared with the wild type following drought treatment.Upregulated genes included those encoding transcription factors,protein kinases,heat shock proteins,and other proteins known to be involved in stress responses.We conclude that GhADF1 reduces the expression of abiotic stress-associated genes in cotton response to drought stress and may be a promising candidate gene for crop improvement by genetic manipulation.

Arabidopsis AtADF1 is Functionally Affected by Mutations on Actin Binding Sites

The plant actin depolymerizing factor （ADF） binds to both monomeric and filamentous actin, and is directly involved in the depolymerization of actin filaments. To better understand the actin binding sites of the Arabidopsis thaliana L. AtADF1, we generated mutants of AtADF1 and investigated their functions in vitro and in vivo. Analysis of mutants harboring amino acid substitutions revealed that charged residues （Arg98 and Lys100） located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G- and F-actin binding. The basic residues on the β-strand 5 （K82/A） and the α-helix 4 （R135/A, R137/A） form another actin binding site that is important for F-actin binding. Using transient expression of CFP-tagged AtADF1 mutant proteins in onion （Allium cepa） peel epidermal cells and transgenic Arabidopsis thaliana L. plants overexpressing these mutants, we analyzed how these mutant proteins regulate actin organization and affect seedling growth. Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional, unless the affinity for actin monomers is also affected. The G-actin binding activity of the ADF plays an essential role in actin binding, depolymerization of actin polymers, and therefore in the control of actin organization.

Effect of manipulation on cartilage in rats with knee osteoarthritis based on the Rho-associated protein kinase/LIM kinase 1/Cofilin signaling pathways

OBJECTIVE:To investigate the effect of manipulation treatment on knee osteoarthritis rats and the effect on Rho-associated protein kinase(ROCK)/LIM-kinase1(LIMK1)/Cofilin signaling pathway.METHOD:Fifty Specific pathogen Free Sprague-Dawley rats were randomly divided into five groups(n=8 each):blank group,model group,manipulation group,celecoxib group,and manipulation combined with celecoxib group(MC group).The osteoarthritis model was established by injecting 0.2 m L 4%papain into the articular disc of the rats.After successfully establishing the model,we treated the manipulation group with pushing manipulation using one-finger-meditation to the Neixiyan(EX-LE4),Waixiyan(EX-LE5),Xuehai(SP10),Liangqiu(ST34),and Zusanli(ST36)acupoints for 10 min each time.Also,the celecoxib group was gavaged with 24 mg·kg^(-1)·d^(-1 )celecoxib,while the MC group was treated using both of these two methods.After four weeks,the cartilage of the right femur was removed for hematoxylin-eosin staining of the cartilage tissue.The expressions of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in serum were observed using the enzyme-linked immunosorbent assay.Besides,we detected the expressions of ROCK,LIMK1,Phospho-LIM-kinase1(Phospho-LIMK1),Cofilin,and Phospho-Cofilin by Western blot.RESULTS:Compared to the model group,the manipulation group,celecoxib group,and MC group all exhibited superior results concerning pathological morphologic changes of cartilage,as observed by hematoxylin-eosin staining and calculated using the Mankin score.Besides,in contrast to the blank group,the model group exhibited elevated serum levels of IL-1βand TNF-α(P<0.01),while the expression of ROCK,LIMK1,Phospho-LIMK1,Cofilin,and Phospho-Cofilin in cartilage were all higher(P<0.01).Also,the serum levels of IL-1βand TNF-αin each treatment group were lower(P<0.01)than in the model group.Moreover,there were lower expressions of ROCK,LIMK1,Phospho-LIMK1,Cofilin,and Phospho-Cofilin in cartilage in the manipulation group and the MC group(P<0.01).Compared with the model group,the expression of ROCK,LIMK1,PhosphoLIMK1,Cofilin,and Phospho-Cofilin in cartilage in the celecoxib group were not statistically different(P>0.05).CONCLUSION:In this study,we established that manipulation has a better curative effect than celecoxib.Manipulation inhibits the development of cytoskeleton damage in cartilage and slows articular degeneration by regulating the expression of related proteins in the cytoskeletal signaling pathway.