Rice Leaf Tissues Research Articles

Structural changes of mesophyll cells in the rice leaf tissue in response to salinity stress based on the three-dimensional analysis.

Rice leaf blades have intricate-shaped mesophyll cells (MCs) with a large volume of chloroplasts enhancing gas exchange between stroma and intercellular airspace (IAS). Since the rice MCs do not form palisade or spongy tissue cells and are considered monotypic cells, the structural analysis of MCs in the middle part of the leaf tissue has been done, neglecting the various shapes of MCs can be observed on the cross-section of rice leaves. Moreover, the middle MC layer is sandwiched between the upper and lower layers and is more restricted in its demand for light and CO2 entering from the outside. Therefore, the different layers of MCs may differ in their sensitivity to salt stress that causes structural changes in cells. This study aims to elucidate the intra- and extra-cellular structures of MC in different layers of leaf tissue and determine how salinity affects the MC structure in each layer. The mesophyll tissue was divided into adaxial, middle and abaxial layers, and eight MCs and chloroplast regions were selected from each layer and reconstructed into three-dimensional (3D) representations. The whole leaf anatomical and physiological parameters were measured to find the effects of salinity stress on the MC structures. As a result,the 3D analysis of rice leaf tissue revealed the different structures of MCs with greater diversity in the adaxial and abaxial layers than in the middle layer. Salinity stress reduced the size and height of the MCs and coverage of the chloroplast on the cytoplasm periphery of the adaxial and abaxial layers, as well as the chloroplast size of adaxial MCs. Overall,these results reveal the variation of rice MC in leaf tissue and suggest the higher sensitivity to salt stress in the adaxial mesophyll among the layers, which may partly account for the decrease in photosynthetic capacity.

Sewage sludge-derived nutrients and biostimulants stimulate rice leaf photosynthesis and root metabolism to enhance carbohydrate, nitrogen and antioxidants accumulation

The production of high quality liquid nitrogen fertilizer with both nutrient comprehensive and biostimulant properties by alkaline thermal hydrolysis of sewage sludge has shown great potential in agricultural production. However, little is known about the effects of sewage sludge-derived nutrients, and biostimulants (SS-NB) on leaf photosynthesis and root growth in rice. Phenotypic, metabolic and microbial analyses were used to reveal the mechanism of SS-NB on rice. Compared to NF treatment, phenotypic parameters (fresh/dry weight, soluble sugar, amino acid, protein) were increased by SS-NB in rice. SS-NB can enhance the photosynthesis of rice leaves by improving the photoconversion efficiency, chlorophyll content, ATP synthase activity, Rubisco and NADPH production. Meanwhile, SS-NB also increased antioxidant capacity (SOD, POD, CAT and proline) in rice leaf and root tissues. Metabolomics revealed that SS-NB application increased the expression levels of metabolites in root and leaf tissues, including carbohydrate, nitrogen and sulfur metabolism, amino acid metabolism, antioxidants, and phytohormone. Most importantly, the regulation of metabolites in rice root tissues is more sensitive than in leaf tissues, especially to the higher levels of antioxidants and phytohormones (IAA and GA) in rice root tissues. Furthermore, SS-NB increased the abundance of photosynthetic autotrophic, organic acids-degrading and denitrifying functional bacteria in rice roots and recruited plant growth-promoting bacteria (Azospirillum and norank_f_JG30-KF-CM45), while the NF treatment group resulted in an imbalance of the microbial community, leading to the dominance of pathogenic bacteria. The results showed that SS-NB had great application potential in crop growth and stress resistance improvement.

Assessing the Effects of Cadmium Stress on the Growth, Physiological Characteristics, and Metabolic Profiling of Rice (Oryza sativa L.) Using HPLC-QTOF/MS

Cadmium (Cd) pollution is an important environmental problem, as it is easily absorbed by plants and gradually accumulates in the human body through the food chain. This study aimed to elucidate the changes in the metabolic response of the rice cultivar “TanLiangYou215” under Cd stress. Rice was grown in soil culture at 0 (Control), 2 (Low group), and 10 (High group) mg/kg CdCl2 for 90 days. The ultrastructural, Cd content, antioxidant activity, and metabolic changes to the rice in different tissues were analyzed. Phenotypic characterization and ultrastructure showed that the rice roots and leaves were significantly damaged and plant growth was inhibited in the High group, while plant growth was promoted in the Low group. Overall, Cd showed a regularity of “low promotion and high inhibition”. Physiological indices revealed that rice was significantly affected by Cd stress. Compared to the Control, Cd stress resulted in higher antioxidant enzyme activities, and the Low group suffered less oxidative damage than the High group. Metabolomic studies revealed that Cd stress significantly altered the metabolic profiles of rice plants. Rice responded to Cd stress by upregulating amino acids and regulating related pathways, including alanine, aspartate and glutamate metabolism, and arginine and proline metabolism. The significant expression of flavonoids with antioxidant properties helped rice resist the oxidative damage caused by Cd accumulation in the root tissue; Cd stress significantly downregulated glycerophospholipid metabolism in the stem and leaf tissues, which affected the cellular activities in rice stem and leaf tissues. We investigated the effects of Cd stress on ultrastructure, antioxidant activity, and metabolic changes in different tissues of the rice variety TLY215. Moreover, the different tissues of TLY215 can regulate these metabolic pathways to resist Cd stress, which provides valuable insights into the response of TLY215 to different concentrations of Cd.

Evaluating the Differential Response of Transcription Factors in Diploid versus Autotetraploid Rice Leaves Subjected to Diverse Saline-Alkali Stresses.

Saline-alkali stress is a significant abiotic stress factor that impacts plant growth, development, and crop yield. Consistent with the notion that genome-wide replication events can enhance plant stress resistance, autotetraploid rice exhibited a higher level of tolerance to saline-alkali stress than its donor counterparts, which is reflected by differential gene expression between autotetraploid and diploid rice in response to salt, alkali, and saline-alkali stress. In this study, we investigated the expression of the transcription factors (TFs) in the leaf tissues of autotetraploid and diploid rice under different types of saline-alkali stress. Transcriptome analysis identified a total of 1040 genes from 55 TF families that were altered in response to these stresses, with a significantly higher number in autotetraploid rice compared to diploid rice. Contrarily, under these stresses, the number of expressed TF genes in autotetraploid rice was greater than that in diploid rice for all three types of stress. In addition to the different numbers, the differentially expressed TF genes were found to be from significantly distinct TF families between autotetraploid and diploid rice genotypes. The GO enrichment analysis unraveled that all the DEGs were distributed with differentially biological functions in rice, in particular those that were enriched in the pathways of phytohormones and salt resistance, signal transduction, and physiological and biochemical metabolism in autotetraploid rice compared to its diploid counterpart. This may provide useful guidance for studying the biological roles of polyploidization in plant resilience in response to saline-alkali stress.

3-D reconstruction of rice leaf tissue for proper estimation of surface area of mesophyll cells and chloroplasts facing intercellular airspaces from 2-D section images.

The surface area of mesophyll cells (Smes) and chloroplasts (Sc) facing the intercellular airspace (IAS) are important parameters for estimating photosynthetic activity from leaf anatomy. Although Smes and Sc are estimated based on the shape assumption of mesophyll cells (MCs), it is questionable if the assumption is correct for rice MCs with concave-convex surfaces. Therefore, in this study, we establish a reconstruction method for the 3-D representation of the IAS in rice leaf tissue to calculate the actual Smes and Sc with 3-D images and to determine the correct shape assumption for the estimation of Smes and Sc based on 2-D section images. We used serial section light microscopy to reconstruct 3-D representations of the IAS, MCs and chloroplasts in rice leaf tissue. Actual Smes and Sc values obtained from the 3-D representation were compared with those estimated from the 2-D images to find the correct shape-specific assumption (oblate or prolate spheroid) in different orientations (longitudinal and transverse sections) using the same leaf sample. The 3-D representation method revealed that volumes of the IAS and MCs accounted for 30 and 70 % of rice leaf tissue excluding epidermis, respectively, and the volume of chloroplasts accounted for 44 % of MCs. The shape-specific assumption on the sectioning orientation affected the estimation of Smes and Sc using 2-D section images with discrepancies of 10-38 %. The 3-D representation of rice leaf tissue was successfully reconstructed using serial section light microscopy and suggested that estimation of Smes and Sc of the rice leaf is more accurate using longitudinal sections with MCs assumed as oblate spheroids than using transverse sections with MCs as prolate spheroids.

Enhanced photoactivity of Bi2S3 nanoflowers by CS-AgBr and CeO2: Application in photoelectrochemical biosensor for the effect of antibiotics on N6-methyladenosine in rice tissues

• The CS-AgBr greatly improves the photoelectric activity of Bi 2 S 3 . • N 6 -methyladenosine-5′-triphosphate was specifically recognized and detected by PEC biosensor. • The photocurrent response is enhanced by the band matching of Bi 2 S 3 /CS-AgBr/CeO 2 . • The effect of three antibiotics on m 6 ATP expression in rice seedlings was investigated. CS-AgBr and octahedral CeO 2 greatly enhanced the photoactivity of Bi 2 S 3 nanoflowers due to the Bi 2 S 3 can form an energy band match with CS-AgBr and CeO 2 , which can accelerate electron transfer and reduce photogenerated electron-hole pair recombination. Thus, CeO 2 /CS-AgBr/Bi 2 S 3 exhibits good photoelectrochemical (PEC) response. With this as a basis, a novel PEC immunosensor was intended for N 6 -methyladenosine-5′-triphosphate (m 6 ATP) detection, in which m 6 ATP antibody was applied as the recognition unit for m 6 ATP, Bi 2 S 3 /CS-AgBr was utilized towards the photoactive material, and CeO 2 was employed as the signal amplification unit. m 6 ATP antibody was firstly captured on CPBA/CS-AgBr/Bi 2 S 3 /ITO surface through the covalent reaction between the boronic acid group of 4-carboxyphenylboronic acid (CPBA) and the o-diol structure of m 6 ATP antibody. Then, m 6 ATP was specifically recognized based on the specific immune reaction between the antigen and the antibody. Finally, signaling amplification unit of CeO 2 achieved signal enhancement through the capture of the phosphate group of m 6 ATP. Under optimal conditions, the PEC biosensor exhibited a wide linear range of 0.001–150 nM and a low detection limit of 0.48 pM (3σ). The suitability was assessed by investigating the results of the effect of antibiotics on the m 6 A levels in total RNA of rice root and leaf tissues. The results indicated that all of the three antibiotics (tobramycin, amoxicillin and chloramphenicol) inhibited the expression of m 6 ATP within 1, 4 and 7 days of cultivation.

In vitro and in silico studies of salicylic acid on systemic induced resistance against bacterial leaf blight disease and enhancement of crop yield

Salicylic acid (SA) is an effective elicitor to promote plant defenses and growth. This study aimed to investigate rice (Oryza sativa L.) cv. Khao Dawk Mali 105 treated with salicylic acid (SA)-Ricemate as an enhanced plant protection mechanism against bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo). Results indicated that the use of SA-Ricemate as a foliar spray at concentrations of more than 100 mg L−1 can reduce the severity of BLB disease by 71%. SA-Ricemate treatment also increased the hydrogen peroxide (H2O2) content of rice leaf tissues over untreated samples by 39–61%. Malondialdehyde (MDA) in rice leaves treated with SA-Ricemate also showed an increase of 50–65% when comparing to non-treated samples. The differential development of these defense compounds was faster and distinct when the SA-Ricemate-treated rice was infected with Xoo, indicating plant-induced resistance. Besides, SA-Ricemate elicitor at a concentration of 50–250 mg L−1 was correlated with a substantial increase in the accumulation of total chlorophyll content at 2.53–2.73 mg g−1 of fresh weight which suggests that plant growth is activated by SA-Ricemate. The catalase- and aldehyde dehydrogenase-binding sites were searched for using the CASTp server, and the findings were compared to the template. Chemsketch was used to design and optimize SA, which was then docked to the catalase and aldehyde dehydrogenase-binding domains of the enzymes using the GOLD 3.0.1 Software. SA is shown in several docked conformations with the enzymes catalase and aldehyde dehydrogenase. All three catalase amino acids (GLN7, VAL27, and GLU38) were discovered to be involved in the creation of a strong hydrogen bond with SA when SA was present. In this mechanism, the aldehyde dehydrogenase amino acids LYS5, HIS6, and ASP2 were all implicated, and these amino acids created strong hydrogen bonds with SA. In field conditions, SA-Ricemate significantly reduced disease severity by 78% and the total grain yield was significantly increased which was an increase of plant height, tiller per hill, and panicle in three field trials during Aug–Nov 2017 and 2018. Therefore, SA-Ricemate can be used as an alternative elicitor on replacing harmful pesticides to control BLB disease with a high potential of increasing rice defenses, growth, and yield components.

The Infectivity Survival and Transmissibility of Rice ragged stunt virus from the Frozen-Infected Rice Leaves by the Brown Planthopper, Nilaparvata lugens Stål

Rice ragged stunt virus, which was transmitted by the brown planthopper (Nilaparvata lugens Stål), was an economically important rice plant pathogen, and caused the yield losses of 10 - 100 % in Thailand and Asian rice cultivation areas. The purpose of this research was to assess the infectivity survival and the transmissibility of Rice ragged stunt virus from the frozen infected rice leaf tissues to the hosts, and the efficiency detection by the indirect enzyme-linked immunosorbent assay on nitrocellulose membranes. The result was shown that rice virus was successfully acquired from the infected rice leaf frozen samples at –20 °C (7 days and 10 months) by the insect vectors, which the reactions can be clearly detected on the nitrocellulose membranes. The efficiencies of the insect vector inoculated, and the rice plant transmitted were 60 - 100 % and 20 - 100 %, and the direct variations with increasing of the viral-minimal latent period at the 3 and 10 days after inoculation, respectively. The maximum efficiency detection of crude sap sample dilutions of both viral hosts in the insect vector and the rice plant were the 1:16 to 1:64, and 1:64 to 1:256, respectively. These new idea transmission methods can be applied for the long-term stability, viability and infectivity of the infected samples, the laboratory protocol development of the cryopreservation techniques for the future study purpose of a plant virus, the viral-resistance breeding process, and the impose rice plant protection policies and strategies in Thailand.
 HIGHLIGHTS
 
 Rice ragged stunt virus (RRSV) causes the most destructive rice plant viral disease in Thailand irrigational rice cultivation
 The brown planthopper (BPH) is an economically important vector and a serious management problem for Thai farmers
 The infected rice leaf samples under the frozen and refrigeration conditions were successful as active reservoirs for the viral cycle and transmission
 The RRSV-minimal latent periods (MLP) of the BPH vector and the rice plant were 3 and 10 days after inoculation
 Understanding of plant-virus-vector interactions can take to improve the ability to insect and rice viral disease control policies and strategies in the agro-ecosystem
 
 GRAPHICAL ABSTRACT

Carbon Nanotube-Mediated Plasmid DNA Delivery in Rice Leaves and Seeds.

CRISPR-Cas gene editing technologies offer the potential to modify crops precisely; however, in vitro plant transformation and regeneration techniques present a bottleneck due to the lengthy and genotype-specific tissue culture process. Ideally, in planta transformation can bypass tissue culture and directly lead to transformed plants, but efficient in planta delivery and transformation remains a challenge. This study investigates transformation methods that have the potential to directly alter germline cells, eliminating the challenge of in vitro plant regeneration. Recent studies have demonstrated that carbon nanotubes (CNTs) loaded with plasmid DNA can diffuse through plant cell walls, facilitating transient expression of foreign genetic elements in plant tissues. To test if this approach is a viable technique for in planta transformation, CNT-mediated plasmid DNA delivery into rice tissues was performed using leaf and excised-embryo infiltration with reporter genes. Quantitative and qualitative data indicate that CNTs facilitate plasmid DNA delivery in rice leaf and embryo tissues, resulting in transient GFP, YFP, and GUS expression. Experiments were also initiated with CRISPR-Cas vectors targeting the phytoene desaturase (PDS) gene for CNT delivery into mature embryos to create heritable genetic edits. Overall, the results suggest that CNT-based delivery of plasmid DNA appears promising for in planta transformation, and further optimization can enable high-throughput gene editing to accelerate functional genomics and crop improvement activities.

Probing in vivo RNA Structure With Optimized DMS-MaPseq in Rice.

RNA transcripts form various secondary and tertiary structures that have a wide range of regulatory functions. Several methods have been developed to profile in vivo RNA secondary structure in prokaryotes and eukaryotes. These methods, such as dimethyl sulfate (DMS) mutational profiling with high-throughput sequencing (DMS-MaPseq), couple small chemical-mediated RNA modifications with next-generation sequencing. DMS-MaPseq, a powerful method for genome-wide and target-specific RNA secondary structure profiling, has been applied in yeast, mammals, Drosophila, and Arabidopsis thaliana, but not in crops. Here, we used DMS-MaPseq to conduct a target-specific and genome-wide profile of in vivo RNA secondary structure in rice (Oryza sativa). The DMS treatment conditions were optimized for rice leaf and root tissues. To increase the sequencing depth and coverage of low-abundance transcripts in genome-wide DMS-MaPseq, we used streptavidin-biotin depletion to reduce the abundance of highly expressed chloroplast transcripts during library construction. The resulting target-specific and genome-wide rice DMS-MaPseq data were of high quality and reproducibility. Furthermore, we used DMS-MaPseq to profile the in vivo RNA secondary structure of an OsmiR399 target region located at 5′UTR of OsPHO2, which participates in rice phosphate homeostasis. An unfolded RNA structure downstream of miRNA target site was observed in predicted in vivo RNA secondary structure, reminiscence of the TAM (Target Adjacent nucleotide Motif) involved in mRNA structure-mediated regulation in miRNA cleavage. Our study optimized DMS-MaPseq for probing in vivo RNA secondary structure in rice, facilitating the study of RNA structure-mediated regulations in crops.

Expression levels of genes involved in metal homeostasis, physiological adaptation, and growth characteristics of rice (Oryza sativa L.) genotypes under Fe and/or Al toxicity.

Acid sulphate soil contains high amounts of iron (Fe) and aluminum (Al), and their contamination has been reported as major problems, especially in rainfed and irrigated lowland paddy fields. Rice is sensitive to Fe and Al grown in acid soil (pH < 5.5), leading to growth inhibition and grain yield loss. The objective of this study was to evaluate Fe and/or Al uptake, translocation, physiological adaptation, metal toxicity, and growth inhibition in rice genotypes grown in acid soil. Fe and Al in the root tissues of all rice genotypes were enriched depending on the exogenous application of either Fe or Al in the soil solution, leading to root growth inhibition, especially in the KDML105 genotype. Expression level of OsYSL1 in KDML105 was increased in relation to metal uptake into root tissues, whereas OsVIT2 was downregulated, leading to Fe (50.3 mg g-1 DW or 13.1 folds over the control) and Al (4.8 mg g-1 DW or 2.2 folds over the control) translocation to leaf tissues. Consequently, leaf greenness (SPAD), net photosynthetic rate (Pn), stomatal conductance (gs), and transpiration rate (E) in the leaf tissues of genotype KDML105 under Fe + Al toxicity significantly declined by 28.4%, 35.3%, 55.6%, and 51.6% over the control, respectively. In Azucena (AZU; Fe/Al tolerant), there was a rapid uptake of Fe and Al by OsYSL1 expression in the root tissues, but a limited secretion into vacuole organelles by OsVIT2, leading to a maintenance of low level of toxicity driven by an enhanced accumulation of glutathione together with downregulation of OsGR expression level. In addition, Fe and Al restrictions in the root tissues of genotype RD35 were evident; therefore, crop stress index (CSI) of Fe + Al-treated plants was the maximum, leading to an inhibition of gs (53.6% over the control) and E (49.0% over the control). Consequently, free proline, total phenolic compounds, and ascorbic acid in the leaf tissues of rice under Fe + Al toxicity significantly increased by 3.2, 1.2, and 1.5 folds over the control, respectively, indicating their functions in non-enzymatic antioxidant defense. Moreover, physiological parameters including leaf temperature (Tleaf) increment, high level of CSI (>0.6), SPAD reduction, photon yield of PSII (ΦPSII) diminution, Pn, gs, and E inhibition in rice genotype IR64 (Fe/Al-sensitive) under Fe + Al treatment were clearly demonstrated as good indicators of metal-induced toxicity. Our results on Fe- and/or Al-tolerant screening to find out the candidate genotypes will contribute to present screening and breeding efforts, which in turn help increase rice production in the Fe/Al-contaminated acid soil under lowland conditions.

Identification and molecular characterization of propionylcholinesterase, a novel pseudocholinesterase in rice

ABSTRACT Cholinesterase is consisting of acetylcholinesterase (AChE) and pseudocholinesterase in vertebrates and invertebrates. AChE gene has been identified in several plant species, while pseudocholinesterase gene has not yet been found in any plant species. In this study, we report that the AChE gene paralog encodes propionylcholinesterase (PChE), a pseudocholinesterase in rice. PChE was found to be located adjacent to AChE (Os07g0586200) on rice chromosome 7 and designated as Os07g0586100. Phylogenetic tree analysis showed a close relationship between rice AChE and PChE. PChE-overexpressing rice had higher hydrolytic activity toward propionylthiocholine than acetylthiocholine and showed extremely low activity against butyrylthiocholine. Therefore, the PChE gene product was characterized as a propionylcholinesterase, a pseudocholinesterase. The rice PChE displayed lower sensitivity to the cholinesterase inhibitor, neostigmine bromide, than electric eel, maize, and rice AChEs. The recombinant PChE functions as a 171 kDa homotetramer. PChE was expressed during the later developmental stage, and it was found be localized in the extracellular spaces of the rice leaf tissue. These results suggest that the rice plant possesses PChE, which functions in the extracellular spaces at a later developmental stage. To the best of our knowledge, this study provides the first direct evidence and molecular characterization of PChE in plants.

Enterobacter asburiae and Pantoea ananatis Causing Rice Bacterial Blight in China.

Rice bacterial blight is a devastating bacterial disease threatening rice yield all over the world and Xanthomonas oryzae pv. oryzae is traditionally believed to be the pathogen. In recent years, we have received diseased rice samples with symptoms of blighted leaves from Sichuan and Guangdong provinces, China. Pathogen isolation and classification identified two different enterobacteria as the causal agents, namely Enterobacter asburiae and Pantoea ananatis. Among them, E. asburiae was isolated from samples of both provinces, and P. ananatis was only isolated from the Sichuan samples. Different from rice foot rot pathogen Dickeya zeae EC1 and rice bacterial blight pathogen X. oryzae pv. oryzae PXO99A, strains SC1, RG1, and SC7 produced rare cell wall degrading enzymes (CWDEs) but more extrapolysaccharides (EPS). E. asburiae strains SC1 and RG1 produced bacteriostatic substances while P. ananatis strain SC7 produced none. Pathogenicity tests indicated that all of them infected monocotyledonous rice and banana seedlings, but not dicotyledonous potato, radish, or cabbage. Moreover, strain RG1 was most virulent, while strains SC1 and SC7 were similarly virulent on rice leaves, even though strain SC1 propagated significantly faster in rice leaf tissues than strain SC7. This study firstly discovered E. asburiae as a new pathogen of rice bacterial blight, and in some cases, P. ananatis could be a companion pathogen. Analysis on production of virulence factors suggested that both pathogens probably employ a different mechanism to infect hosts other than using cell wall degrading enzymes to break through host cell walls.

Multiplex CRISPR/Cas9-mediated genome editing of the FAD2 gene in rice: a model genome editing system for oil palm

BackgroundGenome editing employing the CRISPR/Cas9 system has been widely used and has become a promising tool for plant gene functional studies and crop improvement. However, most of the applied CRISPR/Cas9 systems targeting one locus using a sgRNA resulted in low genome editing efficiency. ResultsHere, we demonstrate the modification of the FAD2 gene in rice using a multiplex sgRNA-CRISPR/Cas9 genome editing system. To test the system’s efficiency for targeting multiple loci in rice, we designed two sgRNAs based on FAD2 gene sequence of the Oryza sativa Japonica rice. We then inserted the validated sgRNAs into a CRISPR/Cas9 basic vector to construct pYLCRISPRCas9PUbi-H:OsFAD2. The vector was then transformed into protoplast cells isolated from rice leaf tissue via PEG-mediated transfection, and rice calli using biolistic transformation. Direct DNA sequencing of PCR products revealed mutations consisting of deletions of the DNA region between the two target sgRNAs. ConclusionThe results suggested that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing system may be useful for crop improvement in monocot species that are recalcitrant to genetic modification, such as oil palm.

Association between microsatellite markers and rice response to the rice blast pathogen Magnaporthe oryzae in selected Zambian rice genotypes

Magnaporthe oryzae, the rice blast disease pathogen is estimated to cause losses of up to 30% in rice production worldwide, yet its impact on rice production in Zambia remains unknown. Symptomatic rice leaf tissue collected from rice fields in Mongu District of Zambia was cultured on oatmeal agar medium and fungal colonies were purified and identified through leaf lesions and conidia using published keys. An M. oryzae isolate from this study was used to challenge twenty-eight selected Zambian rice genotypes to determine their responses to infection with the pathogen estimated by lesion numbers and sizes. Twelve of the genotypes (42.8%) were highly resistant (HR) with 1 to 2 lesions per cm2, 6 (21.4%) were moderately resistant with mean lesion numbers of 3 to 4 per cm2 while 10 (35.7%) were susceptible with lesion numbers above 5 per cm2 of leaf tissue. Polymerase chain reaction (PCR)-amplification of rice genomic DNA extracted from the 28 genotypes using selected microsatellite marker primers revealed a moderate genetic diversity with three of them showing significant genetic diversity. Allelic diversity among the selected genotypes was also confirmed from a calculated mean polymorphic information content of 0.8. A correlation between 8 of the microsatellite markers in some of the rice genotypes and their resistance response to M. oryzae was also established. Results show that some of the analysed rice genotypes have a significant potential for use as a genetic resource in breeding for varieties that are resistant to the rice blast pathogen for Zambian farmers.

Immunolocalization Analysis of C4 Proteins in the Leaf Tissue of Rice.

Immunolocalization analysis is a principal tool to study protein expression and subcellular distribution in plant cells or tissues. In this chapter, we present the method of the preparation of lightly fixed fresh rice leaf tissue for immunolocalization analysis and detection of the protein of interest using fluorescent probes by fluorescent microscopy. This method especially does not need the process of embedding plant materials that saves time and prevents alterations of cellular compounds and structure during sample preparation. Using this method, the C4 rice project compared the expressions of the proteins of interest among C4 model plants, wild-type rice, and transgenic or mutant plants and successfully selected the transgenic plants with the correct location of each protein to create a C4 rice prototype.

A simple high-throughput protocol for the extraction and quantification of inorganic phosphate in rice leaves.

PremisePhosphorus (P) is an essential macronutrient that is often limited in agricultural systems. Determining inorganic phosphate (Pi) contents of plant tissues is crucial for evaluating plant P status. Here, we present a simple, high‐throughput colorimetric microplate technique to measure Pi contents in rice (Oryza sativa) leaf tissues, based on the molybdenum blue reaction.Methods and ResultsWe used a hole puncher to sample small equal areas of leaf tissue for Pi extraction. We removed the leaf grinding and weighing steps, which are time‐consuming and normally required to release Pi from the tissues and to measure the biomass for data normalization, respectively. We showed that the punching method yielded comparable results to the conventional grinding method for two rice cultivars grown under various levels of P supply.ConclusionsCompared with existing techniques, this protocol is more suited to an initial screening, enabling one researcher to determine the Pi contents of thousands of rice leaf samples within a few hours.

Transcriptional expression of Na+ homeostasis-related genes and physiological responses of rice seedlings under salt stress

Being a salt-sensitive crop, the growth, development and productivity of rice is severely affected by saline soil. In the present study, Na+ homeostasis, physiological and morphological responses in both the root and leaf tissues of rice (Oryza sativa) seedlings of backcross introgression lines of KDML105 (KD; −/−) × FL530 (+/+) with or without SKC1 and qSt1b QTLs grown under 200 mM NaCl were investigated. Expression levels of OsHKT1;5 in FL530 (+/+) were very low, causing to low Na+ in both the root and leaf tissues, whereas it was up-regulated in KD (−/−), relating to Na+ enrichment. Expression levels of OsHKT2;1 in rice lines, 221-44 (+/+) and 221-48 (+/+) were down-regulated, thus limiting Na+ xylem upload, and store Na+ in the root tissues. OsSOS1 expression level in the leaves of 221-44 (+/+) and 221-58 (+/−) was up-regulated in relation to Na+ efflux. Na+ secretion into vacuoles via OsNHX1 in the leaf tissues was evidently demonstrated in 221-48 (+/+) as a major defense mechanism adopted by the plants to maintain photosynthetic abilities and growth performances, when plants were subjected to salt stress. In addition, the high Fv/Fm ratio in the leaves of 221-58 (+/−) was associated with increased expression level of OsNHX3, in comparison to KD (−/−) seedlings grown under salt stress (diminution by 22.8% over control). Moreover, reduction on growth characters and physiological parameters, viz., shoot fresh weight, shoot height, number of leaves, leaf area, Pn, and accumulation of Na+ in the roots under salt stress were applied as effective indices; thereby classifying FL530 (+/+) and 221-48 (+/+) as salt-tolerant, and 221-44 (+/+), 221-58 (+/−), KD (−/−), 221-3 (+/−), and 221-54 (−/−) as salt-susceptible.

Three-dimensional anatomy of mesophyll cells in rice leaf tissue by serial section light microscopy

ABSTRACT2D sections of rice (Oryza sativa L.) mesophyll cells are complex and the cell interior contains a large volume of chloroplasts. Furthermore, the structure of chloroplasts changes in response to salt stress. In this study, we used a 3D reconstruction method based on serial section light microscopy to analyze a wide range of structures in leaf tissues and compared the intracellular structure of mesophyll cells of rice under normal and salt-treated conditions. The 3D reconstructed models revealed that mesophyll cells appeared as ellipsoid discs with several lobes around the cell periphery, with the volumes showing no significant difference between control and salt-treated plants. The chloroplast structure in the whole mesophyll cell was altered under salt stress, showing a reduced coverage area, whereas their volumes did not differ between treatments. These findings suggest that 3D reconstruction based on serial light micrographs can reveal the morphology of cells and chloroplasts in plant tissue.

Three-Dimensional Anatomy of the Mesophyll Cells at Different Positions in Rice Leaf Tissue

Three-Dimensional Anatomy of the Mesophyll Cells at Different Positions in Rice Leaf Tissue