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Rice is one of the most important staple foods globally, sustaining over half of the world’s population. However, the sustainability of grain production is increasingly threatened by heat stress, which is intensified by global climate change. Heat stress, characterized by temperatures exceeding crop-specific optimal growth thresholds, significantly impacts the rice yield and quality, particularly during critical reproductive stages. This review synthesizes current research on strategies to mitigate heat stress in rice through genetic and agronomic approaches. It highlights the implementation of advanced genetic tools such as marker-assisted selection (MAS) and genomic selection (GS) to accelerate the breeding of heat-tolerant rice varieties. Additionally, it discusses sustainable agronomic practices, including adjusting planting dates, optimizing water management, and crop rotation, which enhance resilience to heat stress. The objective of this review is to bridge the gap between research findings and practical agricultural applications, providing a comprehensive resource that guides future research directions and informs policy interventions. This review emphasizes the importance of integrating genetic innovations with traditional and modern farming practices to develop rice varieties that can withstand the adverse effects of heat stress, ensuring food security and agricultural sustainability in the face of climatic challenges.

The increasing global population and climate change threaten food security, with the need for sustenance expected to rise by 85% by 2050. Rice, a crucial staple food for over 50% of the global population, is a major source of calories in underdeveloped and developing countries. However, by the end of the century, over 30% of rice fields will become saline due to soil salinity caused by earthquakes, tsunamis, and rising sea levels. Plants have developed strategies to deal with salt stress, such as ion homeostasis, antioxidant defense mechanisms, and morphological adaptations. Proline, an endogenous osmolyte, is the predominant endogenous osmolyte that accumulates in response to salinity, and its overexpression in rice plants has been observed to increase plant salinity tolerance. Exogenously applied proline has been shown to improve plant salt tolerance by reducing the destructive effect of salinity. Recent research has focused on ionic toxicity, nitrogen fixation, and gene expression related to salt tolerance. Exogenous proline has been shown to improve water potential and leaf content, restoring water usage efficiency. It can also ease growth inhibition in salt-sensitive plants. Exogenously applied proline increases antioxidant activities and enhances plant salinity tolerance. This review examines the role and processes of proline in rice plants under salt stress and its relationship with other tolerance mechanisms.

Abstract 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.

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.

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.

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.

This study focused on the mitigation of the negative impact of salinity stress over time on the growth and development of Phoenix dactylifera plants using compost and/or arbuscular mycorrhizal fungi (AMF). The experiment had eight treatments in a randomized design. The treatments consisted of a control, AMF (native consortium) and compost (produced from green waste). The treatments were applied separately or in combination in the presence of 240 mM NaCl (saline condition) or 0 mM NaCl (non-saline condition) after 10 and 14 months of cultivation. Our results show that saline stress increased toxic ion (sodium and chlorine), proline, soluble sugars and stress marker (H2O2 and MDA) contents. At the same time, it lowered growth traits, mycorrhizal colonization, leaf water potential and nutrients (nitrogen (N), potassium (K), phosphorus (P), calcium (Ca)) and photosynthetic pigment concentrations. The application of compost and AMF individually or in combination alleviated salt-induced effects through mechanisms such as an increase in nutrient absorption (P, N, K and Ca), photosynthetic pigment content, relative water content, stomatal opening, leaf water potential, photosystem II efficiency, organic osmolyte content (proline and soluble sugars) and antioxidant enzyme activities (SOD, APX, CAT and POD) and reduction in lipid peroxidation and H2O2 content. Our results also show that the tolerance strategies of date palm to salinity were progressively improved over time in treated plants and especially in date palms grown in the presence of the two biofertilizers. Between 10 and 14 months of cultivation, growth parameters increased with a significant improvement in nutrient contents, a reduction in the concentrations of toxic ions and stress markers, and regulation of the antioxidant system (antioxidant enzyme activity and osmolyte content) in both leaves and roots. In conclusion, this investigation highlights the effectiveness of dual application of compost and native AMF in mitigating the deleterious effects of salinity on date palm with an improvement in the tolerance over time.

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.

The date palm is a commercially important woody crop and is a good target plant for improving agricultural yields in extreme environments. However, salinity has been the primary abiotic stress complicating its cultivation and damaging its production worldwide. This study investigated the effect of alleviating salt stress on date palm growth and development by using arbuscular mycorrhizal fungi (AMF) and/or compost. The experiment was arranged in a completely randomized design with eight treatments. The treatments comprised control without inoculation or amendment and application of compost (made from green waste) and AMF (an autochthonous consortium) individually or in combination under non-saline (0 mM NaCl) or saline (240 mM NaCl) conditions. Growth, physiological characteristics, nutrient uptake, chlorophyll content, oxidative stress markers, and antioxidant enzyme activities were assessed. Salt stress increased sodium (Na+) and chlorine (Cl−) content, lipid peroxidation and proline, soluble sugar, and H2O2 content. However, it reduced growth parameters, AMF colonization, leaf water potential, nitrogen (N), phosphorus (P), potassium (K+), calcium (Ca2+), and chlorophyll content. The application of AMF and compost separately or in combination mitigated the deleterious effects induced by salinity. AMF inoculation contributed to plant salt tolerance through strategies such as increased nutrient uptake (particularly P and Ca2+), chlorophyll content, relative water content, stomatal conductance, antioxidant enzymatic activities (superoxide dismutase, ascorbate peroxidase, catalase) and by decreasing lipid peroxidation and H2O2 content. Plants grown in soil amended with compost under salt stress showed an improvement particularly in K+ and proline content and a decrease in H2O2 concentration compared to controls under the saline condition. In the presence of NaCl stress, the dual application of the compost and AMF consortium maximized plant growth, stomatal conductance, leaf water potential, all antioxidant enzyme activities and P, K+, N, and Ca2+ uptake as well as proline and soluble sugar content. However, it reduced Na+ and Cl− uptake and oxidative stress marker content. In conclusion, our study suggests that the application of AMF with compost has the potential to improve the tolerance of date palm seedlings to salt stress more than AMF or compost applied separately.

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.

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 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.

High-temperature stress during grain filling hastens the growth rate of endosperm and causes grain chalkiness. Scanning microscopy of chalky areas reveals loosely packed, rounded starch granules with occasional small pits. Intensive investigation of the transcriptome, proteome, and metabolome in developing caryopses under high-temperature stress revealed the downregulation of starch synthesis enzymes and the upregulation of α-amylases. High-temperature ripening may unbalance the synthesis and degradation of starch in the developing endosperm cells. In addition to starches, storage proteins are synthesized, assembled, and stored in developing seeds. Several lines of evidence suggest that redox regulation affects seed maturation, including the accumulation of storage starches and proteins, and thus grain quality. A heat-tolerant cultivar of rice shows a characteristic high expression of superoxide dismutase (SOD). H2O2 produced by SOD under high-temperature stress possibly acts as a signal that rapidly can promote the expression of stress-response proteins. Herein, we will discuss the possible molecular physiology of grain chalking under high-temperature stress.

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.

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.

Chloroplasts, which perform photosynthesis, are one of the most important organelles in green plants and algae. Chloroplasts maintain an independent genome that includes important genes encoding their photosynthetic machinery and various housekeeping functions. Owing to its non-recombinant nature, low mutation rates, and uniparental inheritance, the chloroplast genome (plastome) can give insights into plant evolution and ecology and in the development of biotechnological and breeding applications. However, efficient methods to obtain high-quality chloroplast DNA (cpDNA) are currently not available, impeding powerful sequencing and further functional genomics research. To investigate effects on rice chloroplast genome quality, we compared cpDNA extraction by three extraction protocols: liquid nitrogen coupled with sucrose density gradient centrifugation, high-salt buffer, and Percoll gradient centrifugation. The liquid nitrogen–sucrose gradient method gave a high yield of high-quality cpDNA with reliable purity. The cpDNA isolated by this technique was evaluated, resequenced, and assembled de novo to build a robust framework for genomic and genetic studies. Comparison of this high-purity cpDNA with total DNAs revealed the read coverage of the sequenced regions; next-generation sequencing data showed that the high-quality cpDNA eliminated noise derived from contamination by nuclear and mitochondrial DNA, which frequently occurs in total DNA. The assembly process produced highly accurate, long contigs. We summarize the extent to which this improved method of isolating cpDNA from rice can provide practical progress in overcoming challenges related to chloroplast genomes and in further exploring the development of new sequencing technologies.

Abstract: We investigated the effects of soaking temperature and duration on the germinability of seeds of rice (Oryza sativa L., cv. Koganemochi, Gohyakumangoku, and Koshihikari) that had been stored for a long period. The germinability of the seeds soaked at 5ºC for 5 d was markedly lower than that of seeds soaked at 12ºC for 5 d. The germinability of the seeds soaked at 5ºC for 24 hr was not increased by subsequent soaking at 12ºC for 4 d. On the other hand, the germinability of the seeds soaked either at 12ºC for 24 hr or at 30ºC for 80 min was similar to that of seeds soaked at 12ºC for 5 d, even when followed by treatment at 5ºC. Thus, the soaking temperature during the first 24 hr was most important for the germination of rice seeds that had been stored for a long period. Western blotting analysis revealed characteristic expression patterns of α-amylase isoforms in cultivars correlating with the germinability after soaking at a low-temperature.

Next-generation sequencing (NGS) is a revolutionary advancement allowing large-scale discovery of functional molecular markers that has many applications, including plant breeding. High-quality genomic DNA (gDNA) is a prerequisite for successful NGS library preparation and sequencing; however, few reliable protocols to obtain such plant gDNA exist. A previously reported nuclear pellet (NP) method enables extraction of high-yielding gDNA from fresh leaf tissue of maize (Zea mays L.), but the quality does not meet the stringent requirements of NGS. In this study, we optimized the NP method for whole-genome sequencing of rice (Oryza sativa L.) through the integration of simple purification steps. The optimized NP method relied on initial nucleus enrichment, cell lysis, extraction, and subsequent gDNA purification buffers. The purification steps used proteinase K, RNase A, phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform/isoamyl alcohol (24:1) treatments for protein digestion and RNA, protein, and phenol removal, respectively. Our data suggest that this optimized NP method allowed extraction of consistently high-yielding and high-quality undegraded gDNA without contamination by protein and RNA. Moreover, the extracted gDNA fulfilled the quality metrics of NGS library preparation for the Illumina HiSeq X Ten platform by the TruSeq DNA PCR-Free Library Prep Kit (Illumina). We provide a reliable step-by-step guide to the extraction of high-quality gDNA from fresh leaf tissues of rice for molecular biologists with limited resources.

The viability of the estrogen-receptor (ER)-based chemically inducible gene expression system LexA-VP16-ER (XVE) in combination with the site-specific DNA recombination Cre/loxP system in rice was examined using transgenic plants introduced with a plasmid vector pUH-GFP2 that controls the expression of a green fluorescent protein (GFP) gene. β-estradiol applied to the germinating seeds of the transgenic plants, successfully induced the mRNA expression of the GFP gene. Inducible gene suppression was also tested by replacing the GFP gene by an RNAi cassette; this cassette targeted OsSPS1, a gene encoding sucrose phosphate synthase. When the RNAi plants were treated with the inducer, the transcript levels of OsSPS1 decreased. Concomitantly, the plant length became shorter or the sucrose/starch molar ratio in the leaf blades decreased, suggesting the successful suppression of the target gene. Finally, the utility and remaining problems of this inducible expression system are discussed.

In leaves, it is widely assumed that starch is the end-product of a metabolic pathway exclusively taking place in the chloroplast that (a) involves plastidic phosphoglucomutase (pPGM), ADPglucose (ADPG) pyrophosphorylase (AGP) and starch synthase (SS), and (b) is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase (pPGI). This view also implies that AGP is the sole enzyme producing the starch precursor molecule, ADPG. However, mounting evidence has been compiled pointing to the occurrence of important sources, other than the pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility, in this work two independent laboratories have carried out HPLC-MS/MS analyses of ADPG content in leaves of the near-starchless pgm and aps1 mutants impaired in pPGM and AGP, respectively, and in leaves of double aps1/pgm mutants grown under two different culture conditions. We also measured the ADPG content in wild type (WT) and aps1 leaves expressing in the plastid two different ADPG cleaving enzymes, and in aps1 leaves expressing in the plastid GlgC, a bacterial AGP. Furthermore, we measured the ADPG content in ss3/ss4/aps1 mutants impaired in starch granule initiation and chloroplastic ADPG synthesis. We found that, irrespective of their starch contents, pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC accumulate WT ADPG content. In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more ADPG than WT leaves. The overall data showed that, in Arabidopsis leaves, (a) there are important ADPG biosynthetic pathways, other than the pPGI-pPGM-AGP pathway, (b) pPGM and AGP are not major determinants of intracellular ADPG content, and (c) the contribution of the chloroplastic ADPG pool to the total ADPG pool is low.

The effects of various constant temperatures (4, 9, 14, 19, 24, 29, 34, and 38°C) on the germination of winter wheat seed ( Triticum aestivum L. cv. ‘Koyuki’) in a dark condition were studied. The maximum germination percentage was 98% at 24°C. The speed of germination was fastest at 29°G. These results indicate that the most suitable temperature for germination was in the range of 24 to 29°G. α-Amylase expression during germination was also high at higher temperature, and maximum expression occurred at 29°C, although a high temperature of 38°C prevented the synthesis of α-amylase. The close correlation between germination and α-amylase activity at various temperatures indicates that α-amylase is an essential factor for the temperature-dependent germination of wheat seed. In contrast, accumulation of proline increased at a lower temperature, and was the highest at 4°G. We also studied the effects of gibberellin (GA3) and proline, a compatible osmotica in alleviating the effect of low and high temperature stresses. Pre-soaking treatment with GA3 and proline was effective in promoting germination and increasing α-amylase expression at a low (4°C) and high (38°C) temperature. These results suggest that GA3 and proline exhibit positive effects on stress alleviation through the stimulation of α-amylase expression.

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. [ABSTRACT FROM AUTHOR]