Integration of mRNA and miRNA Analysis Reveals the Post-Transcriptional Regulation of Salt Stress Response in Hemerocallis fulva.

MicroRNAs (miRNAs) belong to non-coding small RNAs which have been shown to take a regulatory function at the posttranscriptional level in plant growth development and response to abiotic stress. Hemerocallis fulva is an herbaceous perennial plant with fleshy roots, wide distribution, and strong adaptability. However, salt stress is one of the most serious abiotic stresses to limit the growth and production of Hemerocallis fulva. To identify the miRNAs and their targets involved in the salt stress resistance, the salt-tolerant H. fulva with and without NaCl treatment were used as materials, and the expression differences of miRNAs-mRNAs related to salt-tolerance were explored and the cleavage sites between miRNAs and targets were also identified by using degradome sequencing technology. In this study, twenty and three significantly differential expression miRNAs (p-value < 0.05) were identified in the roots and leaves of H. fulva separately. Additionally, 12,691 and 1538 differentially expressed genes (DEGs) were also obtained, respectively, in roots and leaves. Moreover, 222 target genes of 61 family miRNAs were validated by degradome sequencing. Among the DE miRNAs, 29 pairs of miRNA targets displayed negatively correlated expression profiles. The qRT-PCR results also showed that the trends of miRNA and DEG expression were consistent with those of RNA-seq. A gene ontology (GO) enrichment analysis of these targets revealed that the calcium ion pathway, oxidative defense response, microtubule cytoskeleton organization, and DNA binding transcription factor responded to NaCl stress. Five miRNAs, miR156, miR160, miR393, miR166, and miR396, and several hub genes, squamosa promoter-binding-like protein (SPL), auxin response factor 12 (ARF), transport inhibitor response 1-like protein (TIR1), calmodulin-like proteins (CML), and growth-regulating factor 4 (GRF4), might play central roles in the regulation of NaCl-responsive genes. These results indicate that non-coding small RNAs and their target genes that are related to phytohormone signaling, Ca2+ signaling, and oxidative defense signaling pathways are involved in H. fulva's response to NaCl stress.

Impact of Salinity on the Energy Transfer between Pigment-Protein Complexes in Photosynthetic Apparatus, Functions of the Oxygen-Evolving Complex and Photochemical Activities of Photosystem II and Photosystem I in Two Paulownia Lines.

The present study shows the effect of salinity on the functions of thylakoid membranes from two hybrid lines of Paulownia: Paulownia tomentosa x fortunei and Paulownia elongate x elongata, grown in a Hoagland solution with two NaCl concentrations (100 and 150 mM) and different exposure times (10 and 25 days). We observed inhibition of the photochemical activities of photosystem I (DCPIH2 → MV) and photosystem II (H2O → BQ) only after the short treatment (10 days) with the higher NaCl concentration. Data also revealed alterations in the energy transfer between pigment-protein complexes (fluorescence emission ratios F735/F685 and F695/F685), the kinetic parameters of the oxygen-evolving reactions (initial S0-S1 state distribution, misses (α), double hits (ß) and blocked centers (SB)). Moreover, the experimental results showed that after prolonged treatment with NaCl Paulownia tomentosa x fortunei adapted to the higher concentration of NaCl (150 mM), while this concentration is lethal for Paulownia elongata x elongata. This study demonstrated the relationship between the salt-induced inhibition of the photochemistry of both photosystems and the salt-induced changes in the energy transfer between the pigment-protein complexes and the alterations in the Mn cluster of the oxygen-evolving complex under salt stress.

Protective Effects of Sodium Nitroprusside on Photosynthetic Performance of Sorghum bicolor L. under Salt Stress.

In this study, the impacts of the foliar application of different sodium nitroprusside (SNP, as a donor of nitric oxide) concentrations (0-300 µM) on two sorghum varieties (Sorghum bicolor L. Albanus and Sorghum bicolor L. Shamal) under salt stress (150 mM NaCl) were investigated. The data revealed that salinity leads to an increase in oxidative stress markers and damage of the membrane integrity, accompanied by a decrease in the chlorophyll content, the open photosystem II (PSII) centers, and the performance indexes (PI ABS and PI total), as well as having an influence on the electron flux reducing photosystem I (PSI) end acceptors (REo/RC). Spraying with SNP alleviated the NaCl toxicity on the photosynthetic functions; the protection was concentration-dependent, and greater in Shamal than in Albanus, i.e., variety specific. Furthermore, the experimental results revealed that the degree of SNP protection under salt stress also depends on the endogenous nitric oxide (NO) amount in leaves, the number of active reaction centers per PSII antenna chlorophylls, the enhanced electron flux reducing end acceptors at the acceptor side of PSI, as well as the stimulation of the cyclic electron transport around PSI. The results showed better protection in both varieties of sorghum for SNP concentrations up to 150 µM, which corresponds to about a 50% increase in the endogenous NO leaf content in comparison to the control plants. Our study provides valuable insight into the molecular mechanisms underlying SNP-induced salt tolerance in sorghum varieties and might be a practical approach to correcting salt intolerance.

Integrated analysis of transcriptome and metabolome reveals molecular mechanisms of salt tolerance in seedlings of upland rice landrace 17SM-19.

Salt stress is a major abiotic stress that threatens global rice production. It is particularly important to improve salt tolerance in upland rice because of its growth environment. Upland rice landrace 17SM-19 with high salt tolerance was obtained from a previous study. In this study, an integrated analysis of transcriptome and metabolome was performed to determine the responses of the rice seedling to salt stress. When treated with 100 mm NaCl, the rice seedling growth was significantly inhibited at 5 d, with inhibition first observed in shoot dry weight (SDW). Changes in potassium (K+) content were associated with changes in SDW. In omics analyses, 1,900 differentially expressed genes (DEGs) and 659 differentially abundant metabolites (DAMs) were identified at 3 d after salt stress (DAS), and 1,738 DEGs and 657 DAMs were identified at 5 DAS. Correlation analyses between DEGs and DAMs were also conducted. The results collectively indicate that salt tolerance of upland rice landrace 17SM-19 seedlings involves many molecular mechanisms, such as those involved with osmotic regulation, ion balance, and scavenging of reactive oxygen species.

Assessment of the Photosynthetic Apparatus Functions by Chlorophyll Fluorescence and P700 Absorbance in C3 and C4 Plants under Physiological Conditions and under Salt Stress.

Functions of the photosynthetic apparatus of C3 (Pisum sativum L.) and C4 (Zea mays L.) plants under physiological conditions and after treatment with different NaCl concentrations (0-200 mM) were investigated using chlorophyll a fluorescence (pulse-amplitude-modulated (PAM) and JIP test) and P700 photooxidation measurement. Data revealed lower density of the photosynthetic structures (RC/CSo), larger relative size of the plastoquinone (PQ) pool (N) and higher electron transport capacity and photosynthetic rate (parameter RFd) in C4 than in C3 plants. Furthermore, the differences were observed between the two studied species in the parameters characterizing the possibility of reduction in the photosystem (PSI) end acceptors (REo/RC, REo/CSo and δRo). Data revealed that NaCl treatment caused a decrease in the density of the photosynthetic structures and relative size of the PQ pool as well as decrease in the electron transport to the PSI end electron acceptors and the probability of their reduction as well as an increase in the thermal dissipation. The effects were stronger in pea than in maize. The enhanced energy losses after high salt treatment in maize were mainly from the increase in the regulated energy losses (ΦNPQ), while in pea from the increase in non-regulated energy losses (ΦNO). The reduction in the electron transport from QA to the PSI end electron acceptors influenced PSI activity. Analysis of the P700 photooxidation and its decay kinetics revealed an influence of two PSI populations in pea after treatment with 150 mM and 200 mM NaCl, while in maize the negligible changes were registered only at 200 mM NaCl. The experimental results clearly show less salt tolerance of pea than maize.

Effect of γ-aminobutyric Acid on Phenolics Metabolism in Barley Seedlings under Low NaCl Treatment.

It has been revealed that high NaCl stress (>60 mmol L-1) induced phenolics accumulation in barley seedlings, with γ-aminobutyric acid (GABA) playing a key role. Interestingly, low NaCl stimulus (20 mmol L-1) enhancing phenolics synthesis and growth of barley seedlings was also reported recently. Hence, exogenous GABA and its bio-synthesis inhibitor 3-mercaptopropionic acid (3-MP) were applied to reveal the mechanism of GABA regulating phenolics metabolism in barley seedlings treated with 20 mmol L-1 NaCl. The contents of total phenolics and flavonoids significantly increased by 11.64% and 14.52% under NaCl, respectively. The addition of GABA further increased phenolics and flavonoids contents, especially for gallic acid, protocatechuic acid, caffeic acid, and quercetin, compared with NaCl treatment. Simultaneously, GABA increased the activities and mRNA levels of phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), and 4-coumalyl CoA ligase (4CL). The addition of 3-MP suppressed the above effects, except for increasing the protein levels of PAL, C4H, and 4CL. Low concentration of NaCl not only promoted growth, but also stimulated endogenous GABA metabolism to affect key enzymes activities and mRNA levels for phenolics synthesis in barley seedlings.

Different Sensitivity Levels of the Photosynthetic Apparatus in Zea mays L. and Sorghum bicolor L. under Salt Stress.

The impacts of different NaCl concentrations (0-250 mM) on the photosynthesis of new hybrid lines of maize (Zea mays L. Kerala) and sorghum (Sorghum bicolor L. Shamal) were investigated. Salt-induced changes in the functions of photosynthetic apparatus were assessed using chlorophyll a fluorescence (PAM and OJIP test) and P700 photooxidation. Greater differences between the studied species in response to salinization were observed at 150 mM and 200 mM NaCl. The data revealed the stronger influence of maize in comparison to sorghum on the amount of closed PSII centers (1-qp) and their efficiency (Φexc), as well as on the effective quantum yield of the photochemical energy conversion of PSII (ΦPSII). Changes in the effective antenna size of PSII (ABS/RC), the electron flux per active reaction center (REo/RC) and the electron transport flux further QA (ETo/RC) were also registered. These changes in primary PSII photochemistry influenced the electron transport rate (ETR) and photosynthetic rate (parameter RFd), with the impacts being stronger in maize than sorghum. Moreover, the lowering of the electron transport rate from QA to the PSI end electron acceptors (REo/RC) and the probability of their reduction (φRo) altered the PSI photochemical activity, which influenced photooxidation of P700 and its decay kinetics. The pigment content and stress markers of oxidative damage were also determined. The data revealed a better salt tolerance of sorghum than maize, associated with the structural alterations in the photosynthetic membranes and the stimulation of the cyclic electron flow around PSI at higher NaCl concentrations. The relationships between the decreased pigment content, increased levels of stress markers and different inhibition levels of the function of both photosystems are discussed.

Cation and Zn Accumulation in Brown Seeds of the Euhalophyte Suaeda salsa Improves Germination Under Saline Conditions.

Salinity inhibits plant growth due to salt ion accumulation in plant cells and reduced absorption of other nutrients such as metal ions; however halophyte plants have evolved mechanisms to survive and thrive in high-salt conditions. The euhalophyte Suaeda salsa generates dimorphic seeds (black and brown), which show marked differences in germination and seedling growth under high-salt conditions. However, it is unclear whether their ionic status differs. Here, to provide insight on the role of ions in salt tolerance, we used inductively coupled plasma mass spectrometry to measure the ion contents in the dimorphic seeds from S. salsa plants treated with or without NaCl. We measured the macroelements Na, K, Mg, and Ca, and the microelements Mn, Fe, Zn, Cu, and Mo. NaCl-treated S. salsa plants produced seeds with significantly reduced metallic element contents and significantly increased Na+ contents. The brown seeds of S. salsa plants treated with 0 and 200 mM NaCl had much higher contents of K+, Ca2+, and Fe2+ compared with the black seeds. However, the S. salsa seeds (both black and brown) from NaCl-treated plants were significantly larger, and had higher germination rate and higher seedling salt tolerance compared with seeds from plants not treated with NaCl. Interestingly, we measured significantly higher Zn2+ contents in the brown seeds from plants treated with NaCl compared with the black seeds. This suggests that the high contents of Zn2+ and other cations affected seed development and salt tolerance during germination under high-salt conditions. These observations provide insight into the mechanisms of salt tolerance in this halophyte and inform efforts to increase salt tolerance in salt-sensitive species.

The photo-inhibition of camphor leaves (Cinnamomum camphora L.) by NaCl stress based on physiological, chloroplast structure and comparative proteomic analysis.

BACKGROUND: The distribution and use of camphor (Cinnamomum camphora L.) trees are constrained by increasing soil salinity in south-eastern China along the Yangtze River. However, the response mechanism of this species to salinity, especially in team of photosynthesis, are unknown. METHODS: Here, we analysed themorphological, physiological, ultrastructural, and proteomic traits of camphor seedlings under NaCl (103.45 mM) treatment in pot experiments for 80 days. RESULTS: The growth was limited because of photosynthetic inhibition, with the most significant disturbance occurring within 50 days. Salinity caused severe reductions in the leaf photosynthetic rate (A n), stomatal conductance (g s), maximal chlorophyll fluorescence (Fm), maximum quantum yield of PSII (Fv/Fm), non-photochemical quenching (NPQ), relative quantum efficiency of PSII photochemistry (ΦPSII), photochemical quenching coefficient (qP) and photo-pigment contents (chlorophyll a (Cha), chlorophyll b (Chb), total chlorophyll (Chl)); weakened the antioxidant effects, including those of malondialdehyde (MDA), superoxide dismutase (SOD) and peroxidase (POD); and injured chloroplasts. The physiologicalresults indicated that the main reason for photo-inhibition was oxidative factors induced by NaCl. The proteomic results based on isobaric tags for relative and absolute quantitation (iTRAQ) further confirmedthat photosynthesis was the most significant disrupted process by salinity (P < 0.01) and there were 30 downregulated differentially expression proteins (DEPs) and one upregulated DEP related to restraint of the photosynthetic system, which affected photosystem I, photosystem II, the Cytochrome b6/f complex, ATP synthase and the light-harvesting chlorophyll protein complex. In addition, 57 DEPs were related to photo-inhibition by redox effect and 6 downregulated DEPs, including O2 evolving complex 33kD family protein (gi-224094610) and five other predicted proteins (gi-743921083, gi-743840443, gi-743885735, gi-743810316 and gi-743881832) were directly affected. This study provides new proteomic information and explains the possible mechanisms of photo-inhibition caused by salinity on C. camphor.

NaCl treatment on physio-biochemical metabolism and phenolics accumulation in barley seedlings.

Phenolics are important secondary metabolites in plants with strong antioxidant effects. Seeds germination and exogenous stimulation could activate endogenous enzymes to enhance the content of phenolic acids and flavonoids. Barley seeds geminated under NaCl (1-20 mM) treatment to evaluate the accumulation of phenolics in this study. Results showed that NaCl treatment significantly enhanced the growth of seedlings, especially bud length. NaCl treatment up-regulated genes and proteins expression of phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL), resulting in the enhancement of their activities. As a result, phenolic acids and flavonoids contents increased by 11.19% and 32.54%, respectively, in which gallic acid, protocatechuic, fisetin, myricetin and quercetin were affected mostly. Moreover, NaCl treatment enhanced 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging capacity. Hence, NaCl stimulated the synthesis of phenolic components via enhancing gene, protein expression and the activity of key enzymes.

NaCl treatment markedly enhanced pollen viability and pollen preservation time of euhalophyte Suaeda salsa via up regulation of pollen development-related genes.

Vegetable growth of halophytes has significantly increased through moderate salinity. However, little is known about the reproductive traits of euhalophytes. Male reproduction is pivotal for fertilization and seed production and sensitive to abiotic stressors. The pollen viability and pollen longevity of Suaeda salsa treated with 0 and 200 mM of NaCl were evaluated. It was revealed that the pollen size of S. salsa treated with NaCl was significantly bigger than that in controls. Furthermore, the pollen viability of S. salsa plants treated with NaCl was also significantly higher than that of control after 8 h of the pollens were collected (from 10 to 27 h). The pollen viability of NaCl-treated plants in the field could be maintained for 8 h (from 07:00 to 15:00) in sunny days, which was 1 h longer than that of control plants (from 07:00 to 14:00). Meanwhile, the pollen preservation time of NaCl-treated plants was 16 h at room temperature, which was 8 h longer than that of control plants. Genes related to pollen development, such as SsPRK3, SsPRK4, and SsLRX, exhibited high expression in the flowers of NaCl-treated plants. This indicated that NaCl markedly improved the pollen viability and preservation time via the increased expression of pollen development-related genes, and this benefits the population establishment of halophytes such as S. salsa in saline regions.

Identification of WRKY Transcription Factors Related to Saikosaponin Biosynthesis in Adventitious Roots of Bupleurum chinense / 中草药·英文版

Objective To identify the genes of WRKY transcription factors (TFs) from roots of Bupleurum chinense and genes that potentially regulate saikosaponin (SS) biosynthesis. Methods Firstly, the subfamily cluster analysis was mainly based on Arabidopsis thaliana WRKYs for 27 putative WRKY TFs selected from previous transcriptome sequencing data. Secondly, qPCR was used to screen such genes of WRKY TFs that could be induced by NaCl and PEG6000 in adventitious roots of B. chinense. Meanwhile, saikosaponins (SSs) in treated adventitious roots were determined by HPLC. The roots were collected at 0, 2, 4, 8, 12, 24, 48, and 72 h after treatments, and 120 h only for PEG. Finally, the tissue-specific expression was analyzed on screened genes by qPCR. Results The 27 genes were grouped into three categories: There were nine in Group I, 15 in Group II, and two in Group III. Four genes of WRKYTFs, BCWRKY6, BCWRKY16, BCWRKY32, and BCWRKY35 were obviously induced by NaCl in adventitious roots of B. chinense, while only BCWRKY32 was induced by PEG. The content of SSs increased at different levels in NaCl and PEG6000 treatment. Three genes including BCWRKY6, BCWRKY32, and BCWRKY35, expressed most in roots, were similar to the accumulation pattern of SS. Conclusion The three WRKY genes, BCWRKY6, BCWRKY32, and BCWRKY35, may be involved in the biosynthesis of SS.