The ß-ketoacyl-CoA synthase KCS13 regulates the cold response in cotton by modulating lipid and oxylipin biosynthesis.

Cold stress is a key environmental factor that affects plant development and productivity. In this study, RNA-seq in cotton following cold-stress treatment resulted in the identification of 5239 differentially expressed genes (DEGs) between two cultivars with differing sensitivity to low temperatures, among which GhKCS13 was found to be involved in the response. Transgenic plants overexpressing GhKCS13 showed increased sensitivity to cold stress. KEGG analysis of 418 DEGs in both GhKCS13-overexpressing and RNAi lines after treatment at 4 °C indicated that lipid biosynthesis and linoleic acid metabolism were related to cold stress. ESI-MS/MS analysis showed that overexpression of GhKCS13 led to modifications in the composition of sphingolipids and glycerolipids in the leaves, which might alter the fluidity of the cell membrane under cold conditions. In particular, differences in levels of jasmonic acid (JA) in GhKCS13 transgenic lines suggested that, together with lysophospholipids, it might mediate the cold-stress response. Our results suggest that overexpression of GhKCS13 probably causes remodeling of lipids in the endoplasmic reticulum and biosynthesis of lipid-derived JA in chloroplasts, which might account for the increased sensitivity to cold stress in the transgenic plants. Complex interactions between lipid components, lipid signaling molecules, and JA appear to determine the response to cold stress in cotton.

Fibre elongation requires normal redox homeostasis modulated by cytosolic ascorbate peroxidase in cotton (Gossypium hirsutum).

High-quality cotton fibre equates to a more comfortable textile. Fibre length is an important index of fibre quality. Hydrogen peroxide (H2O2) acts as a signalling molecule in the regulation of fibre elongation. Results from in vitro ovule culture suggest that the alteration of fibre cell H2O2 levels affects fibre development. Ascorbate peroxidase (APX) is an important reactive oxygen species (ROS) scavenging enzyme, and we found that GhAPX1AT/DT encoded one member of the previously unrealized group of cytosolic APXs (cAPXs) that were preferentially expressed during the fibre elongation stage. Transgenic cottons with up- and down-regulation of GhAPX1AT/DT were generated to control fibre endogenous levels of H2O2 Suppression of all cAPX (IAO) resulted in a 3.5-fold increase in H2O2 level in fibres and oxidative stress, which significantly suppressed fibre elongation. The fibre length of transgenic lines with over-expression or specific down-regulation of GhAPX1AT/DT did not show any obvious change. However, the fibres in the over-expression lines exhibited higher tolerance to oxidative stress. Differentially expressed genes (DEGs) in fibres at 10 days post-anthesis (DPA) of IAO lines identified by RNA-seq were related to redox homeostasis, signalling pathways, stress responses and cell wall synthesis, and the DEGs that were up-regulated in IAO lines were also up-regulated in the 10 DPA and 20 DPA fibres of wild cotton compared with domesticated cotton. These results suggest that optimal H2O2 levels and redox state regulated by cytosolic APX are key mechanisms regulating fibre elongation, and dysregulation of the increase in H2O2 induces oxidative stress and results in shorter fibres by initiating secondary cell wall-related gene expression.

Cytosolic Ascorbate Peroxidases Plays a Critical Role in Photosynthesis by Modulating Reactive Oxygen Species Level in Stomatal Guard Cell.

Photosynthetic rate is one of the key factors limiting yield of cotton. Reactive oxygen species (ROS) generated by abiotic stress imposes numerous detrimental effects and causes tremendous loss of yield. It is worth to study whether ROS scavenging enzymes could affect yield through regulating photosynthetic rate in cotton. In this study, we created transgenic cotton with changes of endogenous ROS by overexpressing or suppressing the expression of cytosolic ascorbate peroxidases (APXs), which are hydrogen peroxide (H2O2) scavenging enzymes in plants. The suppression of cytosolic APXs by RNAi brings about a great influence on plant growth and development. Plant height and leaf size declined, and yield-related traits including single boll weight, seed weight, seed size, and lint weight dropped significantly, in IAO lines (cytosolic APX-suppressed lines). The stunted plant growth was due to the decrease of plant photosynthetic rate. The evidences showed that increased ROS level in guard cells inhibited stomatal opening and suppressed the absorption of CO2 and H2O in IAO line. The decrease of water content and the increase of water loss rate in leaf exacerbated the decline of photosynthetic rate in cytosolic APX-suppressed lines. Based on these results, it implies that cytosolic APXs as a whole play an important role in maintaining REDOX balance to regulate photosynthetic rate and yield in cotton.

Polyphosphoester-Camptothecin Prodrug with Reduction-Response Prepared via Michael Addition Polymerization and Click Reaction.

Polyphosphoesters (PPEs), as potential candidates for biocompatible and biodegradable polymers, play an important role in material science. Various synthetic methods have been employed in the preparation of PPEs such as polycondensation, polyaddition, ring-opening polymerization, and olefin metathesis polymerization. In this study, a series of linear PPEs has been prepared via one-step Michael addition polymerization. Subsequently, camptothecin (CPT) derivatives containing disulfide bonds and azido groups were linked onto the side chain of the PPE through Cu(I)-catalyzed azidealkyne cyclo-addition "click" chemistry to yield a reduction-responsive polymeric prodrug P(EAEP-PPA)-g-ss-CPT. The chemical structures were characterized by nuclear magnetic resonance spectroscopy, gel permeation chromatography, Fourier transform infrared, ultraviolet-visible spectrophotometer, and high performance liquid chromatograph analyses, respectively. The amphiphilic prodrug could self-assemble into micelles in aqueous solution. The average particle size and morphology of the prodrug micelles were measured by dynamic light scattering and transmission electron microscopy, respectively. The results of size change under different conditions indicate that the micelles possess a favorable stability in physiological conditions and can be degraded in reductive medium. Moreover, the studies of in vitro drug release behavior confirm the reduction-responsive degradation of the prodrug micelles. A methyl thiazolyl tetrazolium assay verifies the good biocompatibility of P(EAEP-PPA) not only for normal cells, but also for tumor cells. The results of cytotoxicity and the intracellular uptake about prodrug micelles further demonstrate that the prodrug micelles can efficiently release CPT into 4T1 or HepG2 cells to inhibit the cell proliferation. All these results show that the polyphosphoester-based prodrug can be used for triggered drug delivery system in cancer treatment.

Dual-responsive core-crosslinked polyphosphoester-based nanoparticles for pH/redox-triggered anticancer drug delivery.

"Intelligent" crosslinked nanoparticles (NPs) provide great advantages in enhancing drug bioavailability and reducing side effects in anticancer therapeutics. In this study, a novel biodegradable polyphosphoester-based functional copolymer prodrug PTX-(PBYP-g-MPA)-b-PEEP was prepared to construct pH/redox dual-responsive core-crosslinked nanoparticles (DOX/CCL NPs), in which paclitaxel (PTX) was conjugated to the polyphosphoester to form an amphiphilic prodrug and doxorubicin (DOX) was encapsulated inside the prodrug NPs. At first, PTX was used as an initiator to polymerize 2-(but-3-yn-1-yloxy)-2-oxo-1,3,2-dioxaphospholane (BYP) and 2-ethoxy-2-oxo-1,3,2-dioxaphospholane (EOP) by one-pot sequential ring-opening polymerization, yielding a biodegradable polymeric prodrug PTX-PBYP-b-PEEP. Subsequently, a radical-mediated thiol-yne "click" reaction was performed between the alkynyl groups on the PBYP segment and the thiol group of 3-mercaptopropionic acid (MPA) to form a functional carboxyl group at the side chain. The potential positively charged DOX·HCl can be physically encapsulated via electrostatic interaction with the carboxyl group and hydrophobic interaction. Afterwards, the DOX/CCL NPs with cleavable disulfide (S-S) linkages can be formed by partial crosslinking through amidation between the pendant carboxyl groups and cystamine. These NPs possess multifunctional characteristics used for in vitro drug release. Notably, a redox-responsive crosslinker, cystamine dihydrochloride, and synergetic non-covalent interactions not only stabilize the nanoparticles, achieve high DOX-loading capacity of drug loading content (DLC, 14.6%) and drug loading efficiency (DLE, 73.1%), but also endow the DOX/CCL NPs with controlled drug release capacity, which is due to the cleavage of S-S bonds in the presence of 10 mM glutathione (GSH) and weakened electrostatic interaction caused by the protonation of carboxyl groups at a lower pH (5.0). Moreover, these pH/redox dual-responsive DOX/CCL NPs can be steadily internalized by HeLa cells, exhibiting high-efficiency cellular proliferation inhibition. This study presents a promising strategy for controlled intracellular drug release in cancer therapy.

Ascorbate Alleviates Fe Deficiency-Induced Stress in Cotton (Gossypium hirsutum) by Modulating ABA Levels.

Fe deficiency causes significant losses to crop productivity and quality. To understand better the mechanisms of plant responses to Fe deficiency, we used an in vitro cotton ovule culture system. We found that Fe deficiency suppressed the development of ovules and fibers, and led to tissue browning. RNA-seq analysis showed that the myo-inositol and galacturonic acid pathways were activated and cytosolic APX (ascorbate peroxidase) was suppressed in Fe-deficient treated fibers, which increased ASC (ascorbate) concentrations to prevent tissue browning. Suppression of cytosolic APX by RNAi in cotton increased ASC contents and delayed tissue browning by maintaining ferric reduction activity under Fe-deficient conditions. Meanwhile, APX RNAi line also exhibited the activation of expression of iron-regulated transporter (IRT1) and ferric reductase-oxidase2 (FRO2) to adapt to Fe deficiency. Abscisic acid (ABA) levels were significantly decreased in Fe-deficient treated ovules and fibers, while the upregulated expression of ABA biosynthesis genes and suppression of ABA degradation genes in Fe-deficient ovules slowed down the decreased of ABA in cytosolic APX suppressed lines to delay the tissue browning. Moreover, the application of ABA in Fe-deficient medium suppressed the development of tissue browning and completely restored the ferric reduction activity. In addition, ABA 8'-hydroxylase gene (GhABAH1) overexpressed cotton has a decreased level of ABA and shows more sensitivity to Fe deficiency. Based on the results, we speculate that ASC could improve the tolerance to Fe deficiency through activating Fe uptake and maintaining ABA levels in cotton ovules and fibers, which in turn reduces symptom formation.