| Early senescence denotes a unique agricultural attribute which impacts economic yield and grain quality in food and feed industry/cultivation,and it arises through several activities regulated by genes,hormones and stress.Unravelling of the essential regulatory mechanisms that underlie senescence may have a substantial impact on increasing future food production.This study was conducted to shed light on the underlying mechanisms involved in the regulation of senescence in rice(Oryza sativa L.).To achieve this aim field and laboratory experiments were conducted.The first part of the study was conducted to evaluate the effects of vacuolar H+-ATPase(VHA-A1)mutated gene on physiological traits of rice isogenic lines.Moreover,laboratory experiment was conducted to test the effect of VHA-A1 mutated gene on the expression of root proteins at grain-filling stage using a label-free proteomic technique.Lastly the influence of vacuolar H+-ATPase(VHA-A1)mutated gene on rhizosphere fungal community using pyrosequencing approach was tested.The physiological data revealed that early senescence induced a reduction in all the measured parameters except for lipid peroxidation,electrolyte leakage and transpiration rate(Tr).Correlation analysis on yield,yield related traits and physiological traits revealed an inverse relationship between enzyme activities,gaseous exchange traits except transpiration rate,root oxidizability,chlorophyll content,instantaneous water use efficiency.In general,malondialdehyde(MDA),electrolyte leakage and transpiration rate were negatively correlated to all the measured traits.Additionally,Non-invasive Micro-Test Technology(NMT)data revealed higher net fluxes of,K+and H+ and lower Ca2+in esl mutant plants compared to wild type plants,thus highlighting the effect of VHA-A1 gene mutation on membrane damage.Proteomic results showed total of 1665 proteins were identified between the wild type and early senescence leaf(esl)mutant roots,160 of which were differentially expressed,and 148 proteins were down-regulated while 12 were up-regulated.Bioinformatic analysis of the differentially accumulated proteins showed that 18 proteins were enriched in cellular component,11 on biological processes,and one in molecular function.The differentially expressed proteins in roots included those mostly involved in defense response,immune response,tissue development,immune system process,and inflammatory immune effectors,membrane organization,response to molecule of bacteria,cell adhesion,response to wounding,biological adhesion,response to bacteria and locomotion.Likewise,12 proteins from the enriched Kyoto Encyclopedia of Genes and Genomes(KEGG)were retrieved,the databases were related to carbohydrates metabolism.The proteins were enriched in several pathways,including the fructose and mannose,inositol phosphate,glycolysis and others.It is noteworthy to report that important enzymatic proteins involved in carbohydrates and energy metabolism were suppressed in this study.For instance,the 6-Phosphogluconate dehydrogenase(6PGD)is of importance as it is involved in NADPH production,NADPH plays an important role in transport in plants in the form of ATP.However,in the current study this protein was down-regulated which explains the abnormal performance of esl mutant plants relative to their wild type variety.Moreover,four protein pathways were significantly enriched in mutant roots only,namely spliceosome;monobactam biosynthesis;glycine,serine and threonine metabolism;and lysine biosynthesis.This infers that VHA-A1 gene mutation caused the accumulation of these amino acids in roots and therefore we assume they play significant role in assisting plants to cope with the effects of premature senescence.Moreover,stress defense and detoxification proteins were identified such as chloroplastic thioredoxin H.1.This protein has a protective role in roots by preventing the adverse effects of reactive oxygen species(ROS)accumulation thereby saving root cells from early senescence.However,in this study this protein was down-regulated hence why the roots senesced faster in esl mutant than in wild type plants.Again,the expression of heat shock-proteins(Hsp70s)was low,these proteins also have a protective role in protein folding and thus protects cells from stress.Interestingly,the findings of the present study revealed an over-accumulation in the extra cellular matrix(ECM)receptor interaction,ECM is regarded as one strategy developed by plants to defend themselves against harmful effects brought by stress.The network communication between ECM and cells lead to regulation of migration,adhesion,proliferation,differentiation and apoptosis.We therefore assume mutation of VHA-A1 gene triggered the activity of certain enzymes involved in programmed cell death as indicated.Results from pyrosequencing study indicated that early senescence qualities play important roles in defining the overall fungal population and diversity within rice rhizosphere.The most predominate fungal phylum identified within the rhizosphere of the two rice lines belonged to Ascomycota,Basidiomycota and Glomeromycota.While,members from the genera Cladosporium and Thielavia accounted for 4.9%and 4.3%of fungal communities identified in esl and wild-type rhizospheres,respectively.Additionally,the core microbiome in the rhizosphere soils of the tested genotypes contained genera such as Cladosporium,Thielavia,Zopfiella,Gibberella,Taeniolella,Fusarium,Hongkongmyces,Westerdykella,Talaromyces,Phoma,Edenia,Humicola,and Penicillium.Real-time quantitative PCR(RT-PCR)analysis was conducted to verify the pyrosequencing data,the analysis showed a greater abundance of Cladosporium cladosporioides in esl mutant plants than the wild type variety.We therefore surmise that VHA-A1 gene mutation controls ROS which over-accumulate due to reduced detoxification machinery.Over accumulated ROS led to membrane damage as indicated by a higher K+efflux,H+and a lower Ca2+influx observed in esl mutant roots than the wild type variety and reduced root vitality.Additionally,early senescence traits mediated by gene mutation in VHA-A1 led to reduced chlorophyll content thereby a reduced photosynthetic system,hence a decline in biomass accumulation and yield.Again,ROS intern caused oxidative stress in mutant plants which triggered the down-regulation of certain key enzymes involved in energy and carbohydrate metabolism and proteins responsible for protection of plant tissues against adverse effects of ROS stress.These ultimately triggered the up-regulation of the extra cellular matrix(ECM)receptor interaction to switch to programmed cell death(PCD)as one defense strategy.Furthermore,VHA-A1 deletions in mutant plants led to a suppression of a certain group of fungi and abundance of others,such as Cladosporium cladosporioides which typically inhabits decaying and senescing plant matter.Therefore,the over-accumulation of ROS not only caused abnormalities in esl mutant plants,they also protected mutant plants against the adverse effects caused by opportunistic pathogens as indicated by the up-regulated defense and immune response proteins.Identified proteins and metagenomes in this study could become targets for ongoing efforts to improve crops for food and feed industries,thereby ensure food security for all. |
PDF Full Text Request