Exogenous proline suppresses endogenous proline and proline-production genes but improves the salinity tolerance capacity of salt-sensitive rice by stimulating antioxidant mechanisms and photosynthesis.

Salinity is a critical environmental stress factor that significantly reduces crop productivity and yield. A mutant B-type response regulator gene (hst1) has been shown to promote salinity tolerance in the YNU genotype. Previous studies on the hst1 gene showed a higher proline production capacity under salt stress. Using almost identical genetic backgrounded salt-tolerant (YNU) and salt-sensitive (Sister line) rice genotypes, we tested the function of proline in the hst1 gene salinity-tolerance mechanism by applying exogenous proline under control and salt-stress conditions. Morpho-physiological, biochemical, and molecular analysis of ST and SS plants was performed to clarify the salinity tolerance mechanism mediated by the exogenous proline. The ST and SS genotypes accumulated exogenous proline, and the ST genotype has higher proline levels than the SS genotype. However, exogenous proline improved salt tolerance only in the SS genotype. Exogenous proline promotes plant and root growth by stimulating photosynthetic pigments and photosynthesis. The exogenous proline has a reductive effect on MDA, and H2O2 protects plants against ROS. Interestingly, exogenous proline lowers Na+ and raises K+ accumulations under salt stress. In the SS genotype, exogenous proline increases the activity of antioxidant enzymes (SOD, CAT, and APX) to protect against salinity-induced damage. The exogenous proline application down-regulates proline-synthesis genes (OsP5CS1 and OsP5CR) and up-regulates proline-degradation genes. Also, exogenous proline increases the expression of the OsSalT and OsGRAS29 genes, improving salinity tolerance in the SS genotype. Our study has demonstrated that proline plays a significant role in conferring salt tolerance with the salinity-tolerance-related hst1 mechanisms.

Morphological, Molecular Structural and Physicochemical Characterization of Starch Granules Formed in Endosperm of Rice with Ectopic Overexpression of α-Amylase.

The objective of this study was to characterize the endosperm starch in rice that ectopically overexpressed the α-amylase. Transgenic rice plants, transformed with cauliflower mosaic virus 35S promoter driven AmyI-1 (35S::AmyI-1) and AmyII-4 (35S::AmyII-4), and 10 kDa prolamin promoter driven AmyI-1 (P10::AmyI-1), were cultivated under standard conditions (23 °C, 12 h in the dark/ 26 °C, 12 h in the light), and brown grains were subsequently harvested. Each grain displayed characteristic chalkiness, while electron microanalyzer (EPMA)-SEM images disclosed numerous small pits on the surface of the starch granules, attributable to α-amylase activity. Fluorescence labeling and capillary electrophoresis analysis of starch chain length distribution revealed no significant alterations in the starches of 35S::AmyI-1 and 35S::AmyII-4 transgenic rice compared to the wild-type. Conversely, the extremely short α-glucan chains (DP 2-8) exhibited a dramatic increase in the P10::AmyI-1 starch. Rapid visco-analyzer analysis also identified variations in the chain length distribution of P10::AmyI-1 starch, manifesting as changes in viscosity. Moreover, 1H-NMR analysis uncovered dynamic modifications in the molecular structure of starch in rice grain transformed with P10::AmyI-1, which was found to possess unprecedented structural characteristics.

Transgenerational salinity tolerance plasticity in rice: Unraveling nongenetic phenotypic modifications and environmental influences.

Transgenerational plasticity in plants enables rapid adaptation to environmental changes, allowing organisms and their offspring to adapt to the environment without altering their underlying DNA. In this study, we investigated the plasticity of transgenerational salinity tolerance of rice plants using a reciprocal transplant experimental strategy. Our aim was to assess whether nongenetic environment-induced phenotypic modifications and transgenerational salinity affect the salinity tolerance of progeny while excluding nuclear genomic factors for two generations. Using salt-tolerant and salt-sensitive rice genotypes, we observed that the parentally salt-stressed salt-sensitive genotype displayed greater growth performance, photosynthetic activity, yield performance, and transcriptional responses than did the parentally nonstressed salt-sensitive plants under salt stress conditions. Surprisingly, salt stress-exposed salt-tolerant progeny did not exhibit as much salinity tolerance as salt stress-exposed salt-sensitive progeny under salt stress. Our findings indicate that the phenotypes of offspring plants differed based on the environment experienced by their ancestors, resulting in heritable transgenerational phenotypic modifications in salt-sensitive genotypes via maternal effects. These results elucidated the mechanisms underlying transgenerational plasticity in salinity tolerance, providing valuable insights into how plants respond to changing environmental conditions.

The Promising B-Type Response Regulator hst1 Gene Provides Multiple High Temperature and Drought Stress Tolerance in Rice.

High temperatures, drought, and salt stresses severely inhibit plant growth and production due to the effects of climate change. The Arabidopsis ARR1, ARR10, and ARR12 genes were identified as negative salt and drought stress regulators. However, in rice, the tolerance capacity of the hst1 gene, which is orthologous to the ARR1, ARR10, and ARR12 genes, to drought and multiple high temperature and drought stresses remains unknown. At the seedling and reproductive stages, we investigated the drought (DS) high temperature (HT) and multiple high temperature and drought stress (HT+DS) tolerance capacity of the YNU31-2-4 (YNU) genotype, which carries the hst1 gene, and its nearest genomic relative Sister Line (SL), which has a 99% identical genome without the hst1 gene. At the seedling stage, YNU demonstrated greater growth, photosynthesis, antioxidant enzyme activity, and decreased ROS accumulation under multiple HT+DS conditions. The YNU genotype also demonstrated improved yield potential and grain quality due to higher antioxidant enzyme activity and lower ROS generation throughout the reproductive stage under multiple HT+DS settings. Furthermore, for the first time, we discovered that the B-type response regulator hst1 gene controls ROS generation and antioxidant enzyme activities by regulating upstream and downstream genes to overcome yield reduction under multiple high temperatures and drought stress. This insight will help us to better understand the mechanisms of high temperature and drought stress tolerance in rice, as well as the evolution of tolerant crops that can survive increased salinity to provide food security during climate change.

Tolerance with High Yield Potential Is Provided by Lower Na+ Ion Accumulation and Higher Photosynthetic Activity in Tolerant YNU31-2-4 Rice Genotype under Salinity and Multiple Heat and Salinity Stress.

The yield-reduction effect of abiotic stressors such as salinity and heat stresses with the growing world population threatens food security. Although adverse effects of salinity and heat stress on plant growth and production parameters have been documented, in nature, abiotic stresses occur sequentially or simultaneously. In this study, the stress tolerance and yield capacity of Yukinkomai, YNU31-2-4, and YNU SL rice genotypes tested under control (26 °C, 0 mM NaCl), salinity (26 °C, 75 mM NaCl), heat (31 °C, 0 mM NaCl), and heat and salinity (31 °C, 75 mM NaCl) stress combinations at vegetative and reproductive stages with six different scenarios. The results show that salinity and the heat and salinity combination stresses highly reduce plant growth performance and yield capacity. Heat stress during reproduction does not affect the yield but reduces the grain quality. The YNU31-2-4 genotype performs better under heavy salt and heat and salinity stress then the Yukinkomai and YNU SL genotypes. YNU31-2-4 genotypes accumulate less Na+ and more K+ under salt and multiple stresses. In the YNU31-2-4 genotype, low Na+ ion accumulation increases photosynthetic activity and pigment deposition, boosting the yield. Stress lowers the glucose accumulation in dry seeds, but the YNU31-2-4 genotype has a higher glucose accumulation.

B-type response regulator hst1 controls salinity tolerance in rice by regulating transcription factors and antioxidant mechanisms.

Salinity is a serious environmental problem that limits plant yield in almost half of the agricultural fields. The hitomebore salt tolerant 1(hst1) is a mutant B-type response regulator gene that was reported to improve salinity tolerance in the 'YNU31-2-4' (YNU) genotype. The sister line (SL) is salt-sensitive, and the nearest genomic relative of the YNU plant has the OsRR22 gene, which is the non-mutant form of the hst1 gene. Biochemical and comprehensive transcriptome analysis of SL and YNU plants was performed to clarify the salinity tolerance mechanism(s) mediated by the hst1 gene. The hst1 gene reduced Na+ ions, lipid peroxidation, and H2O2 content, and improve proline and antioxidant enzymes activities under salt stress. Various transporter and gene-specific transcriptional regulator genes up-regulated in presence of the hst1 gene under saline conditions, identifying that post-stress transcription factors (OsbHLH056, OsH43, OsGRAS29, and OsMADS1) contributed to improved salinity tolerance in YNU plants. Specifically, OsSalT, miR156, and OsLPT1.16 genes were up-regulated, while upstream (OsHKs and OsHPs) and downstream regulators of the OsRR22 gene were down-regulated in YNU plants under saline conditions. Notably, the transcription factors reprogramming, upstream and downstream genes, indicate that these pathways are transcriptionally regulated by the hst1 gene. The findings of the regulatory role of the hst1 gene on plant transcriptome provide a greater understanding of hst1-mediated salt tolerance in rice plants. This knowledge will contribute to understanding the salinity tolerance mechanisms in rice and the evolution of salt-tolerant crops with the ability to withstand higher salinity to ensure food security during climate change.

The TaGSK1, TaSRG, TaPTF1, and TaP5CS Gene Transcripts Confirm Salinity Tolerance by Increasing Proline Production in Wheat (Triticum aestivum L.).

Salinity is an abiotic stress factor that reduces yield and threatens food security in the world's arid and semi-arid regions. The development of salt-tolerant genotypes is critical for mitigating yield losses, and this journey begins with the identification of sensitive and tolerant plants. Numerous physiologic and molecular markers for detecting salt-tolerant wheat genotypes have been developed. One of them is proline, which has been used for a long time but has received little information about proline-related genes in wheat genotypes. In this study, proline content and the expression levels of proline-related genes (TaPTF1, TaDHN, TaSRG, TaSC, TaPIMP1, TaMIP, TaHKT1;4, TaGSK, TaP5CS, and TaMYB) were examined in sensitive, moderate, and tolerant genotypes under salt stress (0, 50, 150, and 250 mM NaCl) for 0, 12, and 24 h. Our results show that salt stress increased the proline content in all genotypes, but it was found higher in salt-tolerant genotypes than in moderate and sensitive genotypes. The salinity stress increased gene expression levels in salt-tolerant and moderate genotypes. While salt-stress exposure for 12 and 24 h had a substantial effect on gene expression in wheat, TaPTF1, TaPIMP1, TaMIP, TaHKT1;4, and TaMYB genes were considerably upregulated in 24 h. The salt-tolerant genotypes showed a higher positive interaction than a negative interaction. The TaPTF1, TaP5CS, TaGSK1, and TaSRG genes were found to be more selective than the other analyzed genes under salt-stress conditions. Despite each gene's specific function, increasing proline biosynthesis functioned as a common mechanism for separating salt tolerance from sensitivity.

Root Reinforcement Improved Performance, Productivity, and Grain Bioactive Quality of Field-Droughted Quinoa (Chenopodium quinoa).

Modern agriculture is facing multiple and complex challenges and has to produce more food and fiber to feed a growing population. Increasingly volatile weather and more extreme events such as droughts can reduce crop productivity. This implies the need for significant increases in production and the adoption of more efficient and sustainable production methods and adaptation to climate change. A new technological and environment-friendly management technique to improve the tolerance of quinoa grown to maturity is proposed using native microbial biostimulants (arbuscular mycorrhizal fungi; AMF) alone, in the consortium, or in combination with compost (Comp) as an organic matter source under two water treatments (normal irrigation and drought stress (DS)). Compared with controls, growth, grain yield, and all physiological traits under DS were significantly decreased while hydrogen peroxide, malondialdehyde, and antioxidative enzymatic functions were significantly increased. Under DS, biofertilizer application reverted physiological activities to normal levels and potentially strengthened quinoa's adaptability to water shortage as compared to untreated plants. The dual combination yielded a 97% improvement in grain dry weight. Moreover, the effectiveness of microbial and compost biostimulants as a biological tool improves grain quality and limits soil degradation under DS. Elemental concentrations, particularly macronutrients, antioxidant potential (1,1-diphenyl-2-picrylhydrazyl radical scavenging activity), and bioactive compounds (phenol and flavonoid content), were accumulated at higher levels in biofertilizer-treated quinoa grain than in untreated controls. The effects of AMF + Comp on post-harvest soil fertility traits were the most positive, with significant increases in total phosphorus (47%) and organic matter (200%) content under drought conditions. Taken together, our data demonstrate that drought stress strongly influences the physiological traits, yield, and quality of quinoa. Microbial and compost biostimulation could be an effective alternative to ensure greater recovery capability, thereby maintaining relatively high levels of grain production. Our study shows that aboveground stress responses in quinoa can be modulated by signals from the microbial/compost-treated root. Further, quinoa grains are generally of higher nutritive quality when amended and inoculated with AMF as compared to non-inoculated and compost-free plants.

Impact of Single and Combined Salinity and High-Temperature Stresses on Agro-Physiological, Biochemical, and Transcriptional Responses in Rice and Stress-Release.

Here, for the first time, we aimed to identify in rice the key mechanisms and processes underlying tolerance to high-temperature (HT) or salt stress (SS) alone, the co-occurrence of both stresses, and recovery using physiological and biochemical measurements and gene expression analysis. We also investigated whether recovery from the two stressors depended on the relative intensities/relief of each stressor. Wild type ('Yukinkomai') rice plants were found to be more susceptible to salinity or heat applied individually. SS leads to a depletion of cellular water content, higher accumulation of Na+, and alterations in photosynthetic pigments. The stress-tolerant cultivar 'YNU31-2-4' (YNU) displayed a lower Na+/K+ ratio, higher water content in cells and improved photosynthetic traits, antioxidant system, and expression of defence genes. Strikingly, the SS + HT combination provided a significant level of protection to rice plants from the effects of SS alone. The expression pattern of a selected set of genes showed a specific response and dedicated pathways in plants subjected to each of the different stresses, while other genes were explicitly activated when the stresses were combined. Aquaporin genes were activated by SS, while stress-related (P5CS, MSD1, HSPs, and ions transporters) genes were shaped by HT. Hierarchical clustering and principal component analyses showed that several traits exhibited a gradually aggravating effect as plants were exposed to the combined stresses and identified heat as a mitigating factor, clearly separating heat + salt-stressed from salt-non-heat-stressed plants. Furthermore, seedling recovery was far more dependent on the relative intensities of stressors and cultivars, demonstrating the influence of one stressor over another upon stress-release. Taken together, our data show the uniqueness and complexity of the physiological and molecular network modules used by rice plants to respond to single and combined stresses and recovery.

The Native Arbuscular Mycorrhizal Fungi and Vermicompost-Based Organic Amendments Enhance Soil Fertility, Growth Performance, and the Drought Stress Tolerance of Quinoa.

The present study aimed to determine the effects of biostimulants on the physicochemical parameters of the agricultural soil of quinoa under two water regimes and to understand the mode of action of the biostimulants on quinoa for drought adaptation. We investigated the impact of two doses of vermicompost (5 and 10 t/ha) and arbuscular mycorrhizal fungi applied individually, or in joint application, on attenuating the negative impacts of water shortage and improving the agro-physiological and biochemical traits of quinoa, as well as soil fertility, under two water regimes (well-watered and drought stress) in open field conditions. Exposure to drought decreased biomass, leaf water potential, and stomatal conductance, and increased malondialdehyde and hydrogen peroxide content. Mycorrhiza and/or vermicompost promoted plant growth by activating photosynthesis machinery and nutrient assimilation, leading to increased total soluble sugars, proteins, and antioxidant enzyme activities in the leaf and root. After the experiment, the soil's total organic matter, phosphorus, nitrogen, calcium, and soil glomalin content improved by the single or combined application of mycorrhiza and vermicompost. This knowledge suggests that the combination of mycorrhiza and vermicompost regulates the physiological and biochemical processes employed by quinoa in coping with drought and improves the understanding of soil-plant interaction.

Assemblage of indigenous arbuscular mycorrhizal fungi and green waste compost enhance drought stress tolerance in carob (Ceratonia siliqua L.) trees.

In the current study, an eco-friendly management technology to improve young carob (Ceratonia siliqua L.) tree tolerance to water deficit was set up by using single or combined treatments of arbuscular mycorrhizal fungi (AMF) and/or compost (C). Two groups of young carob have been installed: (i) carob cultivated under well-watered conditions (WW; 70% field capacity (FC)) and (ii) where the plants were drought-stressed (DS; 35% FC) during 2, 4, 6, and 8 months. The effect of used biofertilizers on the course of growth, physiological (photosynthetic traits, water status, osmolytes, and mineral content), and biochemical (hydrogen peroxide (H2O2), oxidative damage to lipids (malondialdehyde (MDA), and membrane stability (MS)) traits in response to short- and long-term droughts were assessed. The dual application of AMF and C (C + AMF) boosted growth, physiological and biochemical parameters, and nutrient uptake in carob under WW and DS. After eight months, C + AMF significantly enhanced stomatal conductance by 20%, maximum photochemical efficiency of PSII by 7%, leaf water potential by 23%, chlorophyll and carotenoid by 40%, plant uptake of mineral nutrients (P by 75%, N by 46%, K+ by 35%, and Ca2+ by 40%), concentrations of soluble sugar by 40%, and protein content by 44% than controls under DS conditions. Notably, C + AMF reduced the accumulation of H2O2 and MDA content to a greater degree and increased MS. In contrast, enzyme activities (superoxide dismutase, catalase, peroxidase, and polyphenoloxidase) significantly increased in C + AMF plants under DS. Overall, our findings suggest that the pairing of C + AMF can mediate superior drought tolerance in young carob trees by increasing leaf stomatal conductance, cellular water content, higher solute concentration, and defense response against oxidative damage during the prolonged period of DS.

Development of new high-salt tolerant bread wheat (Triticum aestivum L.) genotypes and insight into the tolerance mechanisms.

The loss of cropland soils, climate change, and population growth are directly affecting the food supply. Given the higher incidence of salinity and extreme events, the cereal performance and yield are substantially hampered. Wheat is forecast to decline over the coming years due to the salinization widespread as one of the oldest and most environmental severe constraints facing global cereal production. To increase salinity tolerance of wheat, in this study, we developed two new salt-tolerant bread wheats, named 'Maycan' and 'Yildiz'. The salinity tolerance of these lines, their parents, and a salt-sensitive cultivar has been tested from measurements of physiological, biochemical, and genes associated with osmotic adjustment/plant tolerance in cultures containing 0 and 150 mM NaCl at the seedling stage. Differential growth reductions to increased salinity were observed in the salt-sensitive cultivar, with those newly developed exhibiting significantly greater root length, growth of shoot and water content as salinity tolerances overall than their parents. 'Maycan' and 'Yildiz' had higher osmoregulator proline content and antioxidants enzyme activities under salinity than the other bread wheat tested. Notably, an important upregulation in the expression of genes related to cellular ion balance, osmolytes accumulation, and abscisic acid was observed in both new wheat germplasms, which may improve salt tolerance. These finding revealed that 'Maycan' and 'Yildiz' exhibit high-salt tolerance at the seedling stage and differing in their tolerance mechanisms to the other tested cultivars, thereby providing an opportunity for their exploitation as modern bread wheats.

Biofertilizers as Strategies to Improve Photosynthetic Apparatus, Growth, and Drought Stress Tolerance in the Date Palm.

Rainfall regimes are expected to shift on a regional scale as the water cycle intensifies in a warmer climate, resulting in greater extremes in dry versus wet conditions. Such changes are having a strong impact on the agro-physiological functioning of plants that scale up to influence interactions between plants and microorganisms and hence ecosystems. In (semi)-arid ecosystems, the date palm (Phoenix dactylifera L.) -an irreplaceable tree- plays important socio-economic roles. In the current study, we implemeted an adapted management program to improve date palm development and its tolerance to water deficit by using single or multiple combinations of exotic and native arbuscular mycorrhizal fungi (AMF1 and AMF2 respectively), and/or selected consortia of plant growth-promoting rhizobacteria (PGPR: B1 and B2), and/or composts from grasses and green waste (C1 and C2, respectively). We analyzed the potential for physiological functioning (photosynthesis, water status, osmolytes, mineral nutrition) to evolve in response to drought since this will be a key indicator of plant resilience in future environments. As result, under water deficit, the selected biofertilizers enhanced plant growth, leaf water potential, and electrical conductivity parameters. Further, the dual-inoculation of AMF/PGPR amended with composts alone or in combination boosted the biomass under water deficit conditions to a greater extent than in non-inoculated and/or non-amended plants. Both single and dual biofertilizers improved physiological parameters by elevating stomatal conductance, photosynthetic pigments (chlorophyll and carotenoids content), and photosynthetic efficiency. The dual inoculation and compost significantly enhanced, especially under drought stress, the concentrations of sugar and protein content, and antioxidant enzymes (polyphenoloxidase and peroxidase) activities as a defense strategy as compared with controls. Under water stress, we demonstrated that phosphorus was improved in the inoculated and amended plants alone or in combination in leaves (AMF2: 807%, AMF1+B2: 657%, AMF2+C1+B2: 500%, AMF2+C2: 478%, AMF1: 423%) and soil (AMF2: 397%, AMF1+B2: 322%, AMF2+C1+B2: 303%, AMF1: 190%, C1: 188%) in comparison with controls under severe water stress conditions. We summarize the extent to which the dual and multiple combinations of microorganisms can overcome challenges related to drought by enhancing plant physiological responses.

Potential of Native Arbuscular Mycorrhizal Fungi, Rhizobia, and/or Green Compost as Alfalfa (Medicago sativa) Enhancers under Salinity.

Salinity is one of the devastating abiotic stresses that cause reductions in agricultural production. The increased salinization affects alfalfa growth, metabolism, and rhizobium capacity for symbiotic N2 fixation negatively. This study was undertaken to investigate the efficiency of green compost (C; made from green waste), arbuscular mycorrhizal fungi (M; field-sourced native consortium), and/or rhizobium (R; a salt-tolerant rhizobium strain) individually or in combination as an effective strategy to improve alfalfa productivity under non-saline and high-saline (120 mM NaCl) conditions. In addition, we aimed to understand the agro-physiological and metabolic basis as well as glomalin content in the soil of biofertilizers-induced salt tolerance in alfalfa. Here, we show that mycorrhizal infection was enhanced after MR inoculation, while C application decreased it significantly. Salinity reduced growth, physiological functioning, and protein concentration, but the antioxidant system has been activated. Application of the selected biofertilizers, especially C alone or combined with M and/or R improved alfalfa tolerance. The tri-combination CMR mitigated the negative effects of high salinity by stimulating plant growth, roots and nodules dry matters, mineral uptake (P, N, and K), antioxidant system, synthesis of compatible solutes, and soil glomalin content, sustaining photosynthesis-related performance and decreasing Na+ and Cl- accumulation, lipid peroxidation, H2O2 content, and electrolyte leakage.

Photosynthesis in a Changing Global Climate: Scaling Up and Scaling Down in Crops.

Photosynthesis is the major process leading to primary production in the Biosphere. There is a total of 7000bn tons of CO2 in the atmosphere and photosynthesis fixes more than 100bn tons annually. The CO2 assimilated by the photosynthetic apparatus is the basis of crop production and, therefore, of animal and human food. This has led to a renewed interest in photosynthesis as a target to increase plant production and there is now increasing evidence showing that the strategy of improving photosynthetic traits can increase plant yield. However, photosynthesis and the photosynthetic apparatus are both conditioned by environmental variables such as water availability, temperature, [CO2], salinity, and ozone. The "omics" revolution has allowed a better understanding of the genetic mechanisms regulating stress responses including the identification of genes and proteins involved in the regulation, acclimation, and adaptation of processes that impact photosynthesis. The development of novel non-destructive high-throughput phenotyping techniques has been important to monitor crop photosynthetic responses to changing environmental conditions. This wealth of data is being incorporated into new modeling algorithms to predict plant growth and development under specific environmental constraints. This review gives a multi-perspective description of the impact of changing environmental conditions on photosynthetic performance and consequently plant growth by briefly highlighting how major technological advances including omics, high-throughput photosynthetic measurements, metabolic engineering, and whole plant photosynthetic modeling have helped to improve our understanding of how the photosynthetic machinery can be modified by different abiotic stresses and thus impact crop production.

iTRAQ-Based Proteomic Analysis of Rice Grains.

Cereal proteins have formed the basis of human diet worldwide, and their level of consumption is expected to increase. The knowledge of the protein composition and variation of the cereal grains is helpful for characterizing cereal varieties and to identify biomarkers for tolerance mechanisms. Grains produce a wide array of proteins, differing under conditions. Quantitative proteomics is a powerful approach allowing the identification of proteins expressed under defined conditions that may contribute understanding the complex biological systems of grains. Isobaric tags for relative and absolute quantitation (iTRAQ) is a mass spectrometry-based quantitative approach allowing, simultaneously, for protein identification and quantification from multiple samples with high coverage. One of the challenges in identifying grains proteins is their relatively high content (~90-95%) of carbohydrate (starch) and low protein (~4-10%) and lipid (~1%) fractions. In this chapter, we present a robust workflow to carry out iTRAQ quantification of the starchy rice grains.

Functional Analysis of Rice Long-Chain Acyl-CoA Synthetase 9 (OsLACS9) in the Chloroplast Envelope Membrane.

The long-chain acyl-CoA synthetases (LACSs) are involved in lipid synthesis, fatty acid catabolism, and the transport of fatty acids between subcellular compartments. These enzymes catalyze the critical reaction of fatty acyl chains to fatty acyl-CoAs for the triacylglycerol biosynthesis used as carbon and energy reserves. In Arabidopsis, LACSs are encoded by a family of nine genes, with LACS9 being the only member located in the chloroplast envelope membrane. However, the comprehensive role of LACS9 and its contribution to plant metabolism have not been explored thoroughly. In this study, we report on the identification and characterization of LACS9 mutants in rice plants. Our results indicate that the loss-of-function mutations in OsLACS9 affect the architecture of internodes resulting in dwarf plants with large starch granules in the chloroplast, showing the suppression of starch degradation. Moreover, the plastid localization of α-amylase I-1 (AmyI-1)-a key enzyme involved in starch breakdown in plastids-was suppressed in the lacs9 mutant line. Immunological and confocal laser scanning microscopy analyses showed that OsLACS9-GFP is located in the chloroplast envelope in green tissue. Microscopic analysis showed that OsLACS9s interact with each other in the plastid envelope membrane. Furthermore, OsLACS9 is also one of the proteins transported to plastids without a transit peptide or involvement of the Toc/Tic complex system. To identify the plastid-targeting signal of OsLACS9, the transient expression and localization of a series of N-terminal truncated OsLACS9-green fluorescent protein (GFP) fusion proteins were examined. Truncation analyses identified the N-terminal 30 amino acid residues to be required for OsLACS9 plastid localization. Overall, the data in this study provide an advanced understanding of the function of OsLACS9 and its role in starch degradation and plant growth.

Arbuscular Mycorrhizal Fungi Mediate Drought Tolerance and Recovery in Two Contrasting Carob (Ceratonia siliqua L.) Ecotypes by Regulating Stomatal, Water Relations, and (In)Organic Adjustments.

Irregular precipitation and drought caused an increase in tree mortality rates in multiple forest biomes with alterations in both ecosystem services and carbon balance. Carob (Ceratonia siliqua) growth and production in arid and semi-arid ecosystems are likely affected by climate change-induced droughts. Understanding the physiological responses of drought-induced early-stage tree death and strategies to enhance drought tolerance and optimize growth will help tree improvement programs. Mycorrhizal inoculation has a pronounced impact on plant growth, water absorption, mineral nutrition, and protection from abiotic stresses. However, a better understanding of these complex interconnected cellular processes and arbuscular mycorrhizal fungi (AMF)-mediated mechanisms regulating drought tolerance in plants will enhance its potential application as an efficient approach for bio-amelioration of stresses. The objectives of this work were to elucidate the different effects of autochthone AMF on inorganic solute and water content uptakes, organic adjustments (sugar and proteins content), leaf gas exchange (stomatal conductance and efficiency of photosystems I and II), and oxidative damage of two contrasting ecotypes of carob seedlings: coastal (southern ecotype (SE)) and in-land (northern ecotype (NE)) under control (C), drought (by cessation of irrigation for 15 days (15D)), and recovery (R) conditions. Our findings showed that AMF promoted growth, nutrient content, and physiological and biochemical parameters in plants of both ecotypes during C, 15D, and R conditions. After four days of recovery, stomatal conductance (gs), the maximum photochemical efficiency of PSII (Fv/Fm), water content, and plant uptake of mineral nutrients (P, K, Na, and Ca) were significantly higher in shoots of mycorrhizal (AM) than non-mycorrhizal (NM) control plants. Consequently, AMF reduced to a greater degree the accumulation of hydrogen peroxide (H2O2) and oxidative damage to lipid (malondialdehyde (MDA)) content in AM than NM plants in NE and SE, after recovery. Altogether, our findings suggest that AMF can play a role in drought resistance of carob trees at an early stage by increasing the inorganic solutes (P, K, Na, and Ca), water content uptake, organic solutes (soluble sugars and protein content), stomatal conductance, and defense response against oxidative damage during re-watering after drought stress.

Recent Advances in Carbon and Nitrogen Metabolism in C3 Plants.

C and N are the most important essential elements constituting organic compounds in plants. The shoots and roots depend on each other by exchanging C and N through the xylem and phloem transport systems. Complex mechanisms regulate C and N metabolism to optimize plant growth, agricultural crop production, and maintenance of the agroecosystem. In this paper, we cover the recent advances in understanding C and N metabolism, regulation, and transport in plants, as well as their underlying molecular mechanisms. Special emphasis is given to the mechanisms of starch metabolism in plastids and the changes in responses to environmental stress that were previously overlooked, since these changes provide an essential store of C that fuels plant metabolism and growth. We present general insights into the system biology approaches that have expanded our understanding of core biological questions related to C and N metabolism. Finally, this review synthesizes recent advances in our understanding of the trade-off concept that links C and N status to the plant's response to microorganisms.

Essential roles of autophagy in metabolic regulation in endosperm development during rice seed maturation.

Autophagy plays crucial roles in the recycling of metabolites, and is involved in many developmental processes. Rice mutants defective in autophagy are male sterile due to immature pollens, indicating its critical role in pollen development. However, physiological roles of autophagy during seed maturation had remained unknown. We here found that seeds of the rice autophagy-deficient mutant Osatg7-1, that produces seeds at a very low frequency in paddy fields, are smaller and show chalky appearance and lower starch content in the endosperm at the mature stage under normal growth condition. We comprehensively analyzed the effects of disruption of autophagy on biochemical properties, proteome and seed quality, and found an abnormal activation of starch degradation pathways including accumulation of α-amylases in the endosperm during seed maturation in Osatg7-1. These results indicate critical involvement of autophagy in metabolic regulation in the endosperm of rice, and provide insights into novel autophagy-mediated regulation of starch metabolism during seed maturation.