Regulation Of Mineral Elements And Redox Status On Fiber Development In Cotton(Gossypium Hirsutum)

Cotton fiber is one of the longest cells in the nature,and high-quality cotton fiber equates to a more comfortable textile.Environmental stresses,such as high temperature,drought,salt stress,diseases and insect pests,mineral deficiency and heavy metal stress so on,causes significant losses to crop productivity and quality.The research on molecular mechanism of the fiber cell elongation responding to environmental stresses is not only the fundamental issue of fiber development,but also an important aspect of study for cotton breeding.In this research,the effects of eleven essential mineral elements on fiber elongation were systematically analyzed using ovule culture system.Combined with transgenic cottons,we specifically investigated the effect of K,Ca or Fe deficiency on the fiber elongation,interacting with the ABA,JA and ROS.Either one deficiency of three elements in ovule culture medium induced ROS accumulation to suppressed fiber elongation.To explore the relationship between ROS and fiber elongation,we generated up-and down-regulation of GhAPX1AT/DT transgenic cottons to control endogenous H₂O₂ levels in the fiber cells.Meanwhile,we also confirmed the positive role of ASA in the ovule adapting to Fe deficiency through maintaining ABA levels.The main results are as follow: 1.The combination of low K and Ca deficiency promotes fiber elongationEleven essential mineral elements including K,Ca,Mg,P,Fe,Mn,Zn,B,Cu,Mo and Co were analyzed in mediums with six concentrations for each element using ovule culture system.In in vitro ovule culture system,low K?2 mM or 27 mM?suppressed fiber elongation,while Ca deficiency induced ovules browning and low Ca?1.5 mM?promoted fiber elongation under sufficient K conditions.However,when Ca deficiency and low K?Ca at 0 mM and K at 27 mM?were simultaneously applied to cultured ovules,longer fibers were produced.Faster fiber elongation under combined low K+ and Ca2+ deficient conditions was due to cell wall loosening by enzymes xyloglucan endotransglycosylase/hydrolase?XTH?and pectin methylesterase?PME?and a higher accumulation of K mediated by CBL-INTERACTING PROTEIN KINASE 6,encoded by GhCIPK6 gene.The delayed ovule and fiber browning and maintenance of fiber growth under low K+-and Ca2+-deficient conditions was found to be due to a partial compensation for Ca deficiency by low K signaling.Low K signaling enhanced ROS scavenging ability,maintained ABA levels and increased JA content,which in turn suppressed the expression of WRKY,MYB and ERF type of transcription factors to alleviate tissue browning and maintain fiber growth.2.H₂O₂ plays an important role on fiber elongationThe mineral deficiency leads ROS accumulation which results in tissue browning and suppressed development.A peak in H₂O₂ content was detected in 15 DPA fibres consistent with the initiation of secondary cell wall synthesis.Results from in vitro ovules culture suggested that ROS status change affected fiber elongation.GhAPX1AT/DT is one member of the expanded cAPX family and showes the highest expression level during the fiber elongation stage?5-10 DPA?.Then,we constructed three kinds of transgenic lines,GhAPX1 overexpressed lines,GhAPX1 specific interference lines and cytosolic APX suppressed lines.Fibres quality of the transgenic cotton harvested from plants grown in the field over two years,the T3 generation in 2013 and the T4 generation in 2014,using HVI?High Volume Instrument?.Suppression of whole cytosolic APX members?T4?in fibres significantly reduced the fibre length?26.06±0.31 mm and 25.71±0.63 mm?by 8.30%-9.54%,compared with the control?28.42±0.67 mm?.For the IAO lines,all fibre quality indexes worsened,including the fiber length,the Uniformity Index?UI?,Elongation?Elg?,Maturity Ratio?MR?and Short Fibre Index?SFI?,compared with the controls.Qualitative and quantitative detection showed that a serious increase of H₂O₂ in cytosolic APX suppressed lines inhibited fiber elongation.Especially,in 10 DPA fibers,H₂O₂ level in cytosolic APX suppressed lines was about 4.5-fold higher than in the controls.RNA-seq revealed that there were 126 DEGs?Differentially expressed genes?identified in 10 DPA fibers of IAO compared with the control.DEGs upregulated in 10 DPA fibers of cytosolic APX suppressed lines were classified into redox homeostasis,signalling pathways,stress responses and cell wall synthesis.Those DEGs were mostly preferentially expressed in the secondary cell wall synthesis stage and expressed higher in the wild upland cotton than in domesticated cotton at 10 DPA or 20 DPA fiber.Therefore,we propose that the suppression of cytosolic APX induced ROS accumulation which resulted in oxidative stress at the 10 DPA fiber.The oxidative stress leads to the earlier expression of secondary cell wall stage genes in the elongation stage and suppresses fiber elongation.3.The cytosolic APX RNAi lines showed higher sensitivity to oxidative stress and higher tolerance to Fe deficiencyTo check whether the alternation of APX expression affect the tolerance of fiber to the environmental stress,We performed treatment with 37°C heat stress or 1 mM H₂O₂ in ovule culture system,and found that cytosolic APX suppressed lines were more sensitive to 37°C treatment and oxidative stress.However,cytosolic APX suppressed line showed higher tolerance to Fe deficiency.Fe deficiency induced ovule and fiber browning after 10 days culture.However,The occurring of browning in cytosolic APX suppressed line was more slowly.And the fresh weight and ferric reduction activity of Fe deficiency treated ovule in cytosolic APX suppressed line was greater than control.ASA improved the tolerance of cytosolic APX suppressed line to iron deficiency.Firstly,the increase of ASA in iron deficiency treated ovules and fibers was greater in cytosolic APX suppressed lines than null and overexpressor.Secondly,the application of ASA,inhibits the iron deficiency induced browning,but the application of H₂O₂ couldn't.Thirdly,ASA restores the fresh weight per ovule and ferric reduction activity under Fe deficiency condition.Further analysis showed that increased ascorbate in cytosolic APX line upregulated maker genes involving in iron uptake,such as FIT1,IRT2,FRO2 and bHLH 92 to enhance the tolerance to Fe deficiency.4.ASA maintains the ABA levels to improve the tolerance to Fe deficeincyABA level was significantly decreased in iron deficiency treated ovule and fiber.However,the decrease of ABA levels was slower in cytosolic APX suppressed line.Meanwhile,the application of ABA inhibits the development of iron deficiency induced browning,and completely restores the level of ferric reduction activity.The reason that ABA decreased more slowly in RNAi lines was due to the activation of ABA biosynthesis and the suppression of ABA degradation.Meanwhile,ABA 8'-hydroxylase gene?GhABAH1?overexpressed cotton has a decrease level of ABA and displays more sensitivity to Fe deficiency than the wild type cotton.Therefore,we speculate that the suppression of cytosolic APX maintains ABA levels to alleviate the iron deficiency induced browning.