The URL1-ROC5-TPL2 transcriptional repressor complex represses the ACL1 gene to modulate leaf rolling in rice.

Moderate leaf rolling is beneficial for leaf erectness and compact plant architecture. However, our understanding regarding the molecular mechanisms of leaf rolling is still limited. Here, we characterized a semi-dominant rice (Oryza sativa L.) mutant upward rolled leaf 1 (Url1) showing adaxially rolled leaves due to a decrease in the number and size of bulliform cells. Map-based cloning revealed that URL1 encodes the homeodomain-leucine zipper (HD-Zip) IV family member RICE OUTERMOST CELL-SPECIFIC 8 (ROC8). A single-base substitution in one of the two conserved complementary motifs unique to the 3'-untranslated region of this family enhanced URL1 mRNA stability and abundance in the Url1 mutant. URL1 (UPWARD ROLLED LEAF1) contains an ethylene-responsive element binding factor-associated amphiphilic repression motif and functions as a transcriptional repressor via interaction with the TOPLESS co-repressor OsTPL2. Rather than homodimerizing, URL1 heterodimerizes with another HD-ZIP IV member ROC5. URL1 could bind directly to the promoter and suppress the expression of abaxially curled leaf 1 (ACL1), a positive regulator of bulliform cell development. Knockout of OsTPL2 or ROC5 or overexpression of ACL1 in the Url1 mutant partially suppressed the leaf-rolling phenotype. Our results reveal a regulatory network whereby a transcriptional repression complex composed of URL1, ROC5, and the transcriptional corepressor TPL2 suppresses the expression of the ACL1 gene, thus modulating bulliform cell development and leaf rolling in rice.

Narrow Leaf21, encoding ribosomal protein RPS3A, controls leaf development in rice.

Leaf morphology influences photosynthesis, transpiration, and ultimately crop yield. However, the molecular mechanism of leaf development is still not fully understood. Here, we identified and characterized the narrow leaf21 (nal21) mutant in rice (Oryza sativa), showing a significant reduction in leaf width, leaf length and plant height, and increased tiller number. Microscopic observation revealed defects in the vascular system and reduced epidermal cell size and number in the nal21 leaf blade. Map-based cloning revealed that NAL21 encodes a ribosomal small subunit protein RPS3A. Ribosome-targeting antibiotics resistance assay and ribosome profiling showed a significant reduction in the free 40S ribosome subunit in the nal21 mutant. The nal21 mutant showed aberrant auxin responses in which multiple auxin response factors (ARFs) harboring upstream open-reading frames (uORFs) in their 5'-untranslated region were repressed at the translational level. The WUSCHEL-related homeobox 3A (OsWOX3A) gene, a key transcription factor involved in leaf blade lateral outgrowth, is also under the translational regulation by RPS3A. Transformation with modified OsARF11, OsARF16, and OsWOX3A genomic DNA (gDNA) lacking uORFs rescued the narrow leaf phenotype of nal21 to a better extent than transformation with their native gDNA, implying that RPS3A could regulate translation of ARFs and WOX3A through uORFs. Our results demonstrate that proper translational regulation of key factors involved in leaf development is essential to maintain normal leaf morphology.

Genome-wide investigation and expression analysis of APETALA-2 transcription factor subfamily reveals its evolution, expansion and regulatory role in abiotic stress responses in Indica Rice (Oryza sativa L. ssp. indica).

Rice is an important cereal crop that serves as staple food for more than half of the world population. Abiotic stresses resulting from changing climatic conditions are continuously threating its yield and production. Genes in APETALA-2 (AP2) family encode transcriptional regulators implicated during regulation of developmental processes and abiotic stress responses but their identification and characterization in indica rice was still missing. In this context, twenty-six genes distributed among eleven chromosomes in Indica rice encoding AP2 transcription-factor subfamily were identified and their diverse haplotypes were studied. Phylogenetic analysis of OsAP2 TF family-members grouped them into three clades indicating conservation of clades among cereals. Segmental duplications were observed to be principal route of evolution, supporting the higher positive selection-pressure, which were estimated to be originated about 10.57 to 56.72 million years ago (MYA). Conserved domain analysis and intron-exon distribution pattern of identified OsAP2s revealed their exclusive distribution among the specific clades of the phylogenetic tree. Moreover, the members of osa-miR172 family were also identified potentially targeting four OsAP2 genes. The real-time quantitative expression profiling of OsAP2s under heat stress conditions in contrasting indica rice genotypes revealed the differential expression pattern of OsAP2s (6 genes up-regulated and 4 genes down-regulated) in stress- and genotype-dependent manner. These findings unveiled the evolutionary pathways of AP2-TF in rice, and can help the functional characterization under developmental and stress responses.

DEGENERATED PANICLE AND PARTIAL STERILITY 1 (DPS1) encodes a cystathionine ß-synthase domain containing protein required for anther cuticle and panicle development in rice.

Degeneration of apical spikelets and reduced panicle fertility are common reasons for low seed-setting rate in rice (Oryza sativa). However, little is known about the underlying molecular mechanisms. Here, we report a novel degenerated panicle and partial sterility 1 (dps1) mutant that showed panicle apical degeneration and reduced fertility in middle spikelets. dps1 plants were characterized by small whitish anthers with altered cuticle morphology and absence of pollen grains. Amounts of cuticular wax and cutin were significantly reduced in dps1 anthers. Panicles of dps1 plants showed an accumulation of reactive oxygen species (ROS), lower antioxidant activity, and increased programmed cell death. Map-based cloning revealed that DPS1 encodes a mitochondrial-localized protein containing a cystathionine ß-synthase domain that showed the highest expression in panicles and anthers. DPS1 physically interacted with mitochondrial thioredoxin proteins Trx1 and Trx20, and it participated in ROS scavenging. Global gene expression analysis in dps1 revealed that biological processes related to fatty acid metabolism and ROS homeostasis were significantly affected, and the expression of key genes involved in wax and cutin biosynthesis were downregulated. These results suggest that DPS1 plays a vital role in regulating ROS homeostasis, anther cuticle formation, and panicle development in rice.

Engineering abiotic stress tolerance via CRISPR/ Cas-mediated genome editing.

Abiotic stresses, including drought, salinity, temperature, and heavy metals, pose a major challenge for crop production and cause substantial yield reduction worldwide. Breeding tolerant cultivars against these abiotic stresses is the most sustainable and eco-friendly approach to cope with this challenge. Advances in genome editing technologies provide new opportunities for crop improvement by employing precision genome engineering for targeted crop traits. However, the selection of the candidate genes is critical for the success of achieving the desired traits. Broadly speaking, these genes could fall into two major categories, structural and regulatory genes. Structural genes encode proteins that provide stress tolerance directly, whereas regulatory genes act indirectly by controlling the expression of other genes involved in different cellular processes. Additionally, cis-regulatory sequences are also vital for achieving stress tolerance. We propose targeting of these regulatory and/or structural genes along with the cis-regulatory sequences via the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system as a robust, efficient, and practical approach for developing crop varieties resilient to climate change. We also discuss the possibility of creating novel quantitative trait loci for abiotic stress tolerance via the CRISPR/Cas-mediated targeting of promoters. It is hoped that these genome editing tools will not only make a significant contribution towards raising novel plant types having tolerance to multiple abiotic stresses but will also aid in public acceptance of these products in years to come. This article is an attempt to critically evaluate the suitability of available tools and the target genes for obtaining plants with improved tolerance to abiotic stresses.

Arabidopsis E3 Ubiquitin Ligases PUB22 and PUB23 Negatively Regulate Drought Tolerance by Targeting ABA Receptor PYL9 for Degradation.

Drought causes osmotic stress and rapidly triggers abscisic acid (ABA) accumulation in plants. The roles of various ABA receptors in drought tolerance and molecular mechanisms regulating ABA receptor stability needs to be elucidated. Here, we report that Arabidopsis plants overexpressing PYL9, one of the 14 pyrabactin resistance (PYR)/pyrabactin resistance-like (PYL)/regulatory component of ABA receptors (RCAR) family ABA receptors, gained drought tolerance trait. Osmotic stress induced accumulation of the PYL9 protein, which was regulated by the 26S proteasome. PYL9 interacted with two highly homologous plant U-box E3 ubiquitin ligases PUB22 and PUB23. In the cell-free degradation assay, the degradation of GST-PYL9 was accelerated in protein extract from plants overexpressing PUB22 but slowed down in protein extract from the pub22 pub23 double mutant. The in vivo decay of Myc-PYL9 was significantly reduced in the pub22 pub23 double mutant as compared with the wild-type. Additionally, PUB22 also interacted with other ABA receptors such as PYL5, PYL7 and PYL8. Considering the improved drought tolerance in the pub22 pub23 double mutant in previous studies, our results suggest that PUB22 and PUB23 negatively regulate drought tolerance in part by facilitating ABA receptors degradation.

CRISPR/Cas9 mediated disruption of Inositol Pentakisphosphate 2-Kinase 1 (TaIPK1) reduces phytic acid and improves iron and zinc accumulation in wheat grains.

Introduction: Phytic acid (PA) is an important antinutrient agent present in cereal grains which reduces the bioavailability of iron and zinc in human body, causing malnutrition. Inositol pentakisphosphate 2- kinase 1 (IPK1) gene has been reported to be an important gene for PA biosynthesis. Objective: A recent genome editing tool CRISPR/Cas9 has been successfully applied to develop biofortified rice by disrupting IPK1 gene, however, it remained a challenge in wheat. The aim of this study was to biofortify wheat using CRISPR/Cas9. Methods: In this study, we isolated 3 TaIPK1 homeologs in wheat designated as TaIPK1.A, TaIPK1.B and TaIPK1.D and found that the expression abundance of TaIPK1.A was stronger in early stages of grain filling. Using CRISPR/Cas9, we have disrupted TaIPK1.A gene in cv. Borlaug-2016 with two guide RNAs targeting the 1st and 2nd exons. Results: We got several genome-edited lines in the T0 generation at frequencies of 12.7% and 10.8%. Sequencing analysis revealed deletion of 1-23 nucleotides and even an addition of 1 nucleotide in various lines. Analysis of the genome-edited lines revealed a significant decrease in the PA content and an increase in iron and zinc accumulation in grains compared with control plants. Conclusion: Our study demonstrates the potential application of CRISPR/Cas9 technique for the rapid generation of biofortified wheat cultivars.

A putative SUBTILISIN-LIKE SERINE PROTEASE 1 (SUBSrP1) regulates anther cuticle biosynthesis and panicle development in rice.

INTRODUCTION: Panicle abortion is a severe physiological defect and causes a reduction in grain yield. OBJECTIVES: In this study, we aim to provide the characterization and functional analysis of a mutant apa1331 (apical panicle abortion1331). METHODS: The isolated mutant from an EMS-mutagenized population was subjected to SSR analysis and Mutmap assay for candidate gene mapping. We performed phenotypic analysis, anthers cross-sections morphology, wax and cutin profiling, biochemical assays and phylogenetic analysis for characterization and evaluation of apa1331. We used CRISPR/Cas9 disruption for functional validation of its candidate gene. Furthermore, comparative RNA-seq and relative expression analysis were performed to get further insights into mechanistic role of the candidate gene. RESULTS: The anthers from the apical spikelets of apa1331 were degenerated, pollen-less and showed defects in cuticle formation. Transverse sections of apa1331 anthers showed defects in post-meiotic microspore development at stage 8-9. Gas Chromatography showed a significant reduction of wax and cutin in anthers of apa1331 compared to Wildtype (WT). Quantification of H2O2 and MDA has indicated the excessive ROS (reactive oxygen species) in apa1331. Trypan blue staining and TUNEL assay revealed cell death and excessive DNA fragmentation in apa1331. Map-based cloning and Mutmap analysis revealed that LOC_Os04g40720, encoding a putative SUBTILISIN-LIKE SERINE PROTEASE (OsSUBSrP1), harbored an SNP (A > G) in apa1331. Phenotypic defects were only seen in apical spikelets due to highest expression of OsSUBSrP1 in upper panicle portion. CRISPR-mediated knock-out lines of OsSUBSrP1 displayed spikelet abortion comparable to apa1331. Global gene expression analysis revealed a significant downregulation of wax and cutin biosynthesis genes. CONCLUSIONS: Our study reports the novel role of SUBSrP1 in anther cuticle biosynthesis by ROS-mediated programmed cell death in rice.

Impact of Pre-Anthesis Drought Stress on Physiology, Yield-Related Traits, and Drought-Responsive Genes in Green Super Rice.

Optimum soil water availability is vital for maximum yield production in rice which is challenged by increasing spells of drought. The reproductive stage drought is among the main limiting factors leading to the drastic reduction in grain yield. The objective of this study was to investigate the molecular and morphophysiological responses of pre-anthesis stage drought stress in green super rice. The study assessed the performance of 26 rice lines under irrigated and drought conditions. Irrigated treatment was allowed to grow normally, while drought stress was imposed for 30 days at the pre-anthesis stage. Three important physiological traits including pollen fertility percentage (PFP), cell membrane stability (CMS), and normalized difference vegetative index (NDVI) were recorded at anthesis stage during the last week of drought stress. Agronomic traits of economic importance including grain yield were recorded at maturity stage. The analysis of variance demonstrated significant variation among the genotypes for most of the studied traits. Correlation and principal component analyses demonstrated highly significant associations of particular agronomic traits with grain yield, and genetic diversity among genotypes, respectively. Our study demonstrated a higher drought tolerance potential of GSR lines compared with local cultivars, mainly by higher pollen viability, plant biomass, CMS, and harvest index under drought. In addition, the molecular basis of drought tolerance in GSR lines was related to upregulation of certain drought-responsive genes including OsSADRI, OsDSM1, OsDT11, but not the DREB genes. Our study identified novel drought-responsive genes (LOC_Os11g36190, LOC_Os12g04500, LOC_Os12g26290, and LOC_Os02g11960) that could be further characterized using reverse genetics to be utilized in molecular breeding for drought tolerance.

Evaluation of Green Super Rice Lines for Agronomic and Physiological Traits under Salinity Stress.

Rice (Oryza sativa) is an important staple food crop worldwide, especially in east and southeast Asia. About one-third of rice cultivated area is under saline soil, either natural saline soils or irrigation with brackish water. Salinity stress is among the devastating abiotic stresses that not only affect rice growth and crop productivity but also limit its cultivation area globally. Plants adopt multiple tolerance mechanisms at the morphological, physiological, and biochemical levels to tackle salinity stress. To identify these tolerance mechanisms, this study was carried out under both a controlled glass house as well as natural saline field conditions using 22 green super rice (GSR) lines along with two local varieties ("IRRI 6 and Kissan Basmati"). Several morpho-physiological and biochemical parameters along with stress-responsive genes were used as evaluation criteria under normal and salinity stress conditions. Correlation and Principal Component Analysis (PCA) suggested that shoot-related parameters and the salt susceptible index (SSI) can be used for the identification of salt-tolerant genotypes. Based on Agglomerative Hierarchical Cluster (AHC) analysis, two saline-tolerant ("S19 and S20") and saline-susceptible ("S3 and S24") lines were selected for further molecular evaluation. Quantitative RT-PCR was performed, and results showed that expression of 1-5-phosphoribosyl -5-5-phosphoribosyl amino methylidene amino imidazole-4-carboxamide isomerase, DNA repair protein recA, and peptide transporter PTR2 related genes were upregulated in salt-tolerant genotypes, suggesting their potential role in salinity tolerance. However, additional validation using reverse genetics approaches will further confirm their specific role in salt tolerance. Identified saline-tolerant lines in this study will be useful genetic resources for future salinity breeding programs.

Thermal Stresses in Maize: Effects and Management Strategies.

Climate change can decrease the global maize productivity and grain quality. Maize crop requires an optimal temperature for better harvest productivity. A suboptimal temperature at any critical stage for a prolonged duration can negatively affect the growth and yield formation processes. This review discusses the negative impact of temperature extremes (high and low temperatures) on the morpho-physiological, biochemical, and nutritional traits of the maize crop. High temperature stress limits pollen viability and silks receptivity, leading to a significant reduction in seed setting and grain yield. Likewise, severe alterations in growth rate, photosynthesis, dry matter accumulation, cellular membranes, and antioxidant enzyme activities under low temperature collectively limit maize productivity. We also discussed various strategies with practical examples to cope with temperature stresses, including cultural practices, exogenous protectants, breeding climate-smart crops, and molecular genomics approaches. We reviewed that identified quantitative trait loci (QTLs) and genes controlling high- and low temperature stress tolerance in maize could be introgressed into otherwise elite cultivars to develop stress-tolerant cultivars. Genome editing has become a key tool for developing climate-resilient crops. Moreover, challenges to maize crop improvement such as lack of adequate resources for breeding in poor countries, poor communication among the scientists of developing and developed countries, problems in germplasm exchange, and high cost of advanced high-throughput phenotyping systems are discussed. In the end, future perspectives for maize improvement are discussed, which briefly include new breeding technologies such as transgene-free clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas)-mediated genome editing for thermo-stress tolerance in maize.

Agronomic, physiological and molecular characterisation of rice mutants revealed the key role of reactive oxygen species and catalase in high-temperature stress tolerance.

Climatic variations have increased the occurrence of heat stress during critical growth stages, which negatively affects grain yield in rice. Plants adapt to harsh environments, and particularly high-temperature stress, by regulating their physiological and biochemical processes, which are key tolerance mechanisms. The identification of heat-tolerant rice genotypes and reliable selection indices are crucial for rice improvement programs. Here, we evaluated the response of a rice mutant population for high-temperature stress at the seedling and reproductive stages based on agronomic, physiological and molecular indices. Estimates of variance components revealed significant differences (P < 0.001) among genotypes, treatments and their interactions for almost all traits. The principal component analysis showed significant diversity among genotypes and traits under high-temperature stress. The mutant HTT-121 was identified as the most heat-tolerant mutant with higher grain yield, panicle fertility, cell membrane thermo-stability (CMTS) and antioxidant enzyme levels under heat stress. Various seedling-based morpho-physiological traits (leaf fresh weight, relative water contents, malondialdehyde, CMTS) and biochemical traits (superoxide dismutase, catalase and hydrogen peroxide) explained variations in grain yield that could be used as selection indices for heat tolerance in rice during early growth. Notably, heat-sensitive mutants accumulated reactive oxygen species, reduced catalase activity and upregulated OsSRFP1 expression under heat stress, suggesting their key roles in regulating heat tolerance in rice. The heat-tolerant mutants identified in this study could be used in breeding programs and to develop mapping populations to unravel the underlying genetic architecture for heat-stress adaptability.

MINI SEED 2 (MIS2) Encodes a Receptor-like Kinase that Controls Grain Size and Shape in Rice.

BACKGROUND: Grain size is a key agronomic trait that is directly associated with grain yield in rice. Although several genes related to grain size in rice have been identified, our understanding of the mechanism of grain development is still limited. RESULTS: In this study, we reported the characterization of a novel seed size mutant mini seed 2 (mis2), in which the grain showed reduced length, width and thickness along with wrinkled surface. Microscopic analysis revealed that the spikelet epidermal cell size was reduced but the cell number was increased in the mis2 mutant, suggesting that MIS2 controls grain size by coordinately regulating epidermal cell size and cell number. Map-based cloning revealed that MIS2 encodes a receptor-like kinase CRINKLY4 (CR4) which showed the highest expression in developing panicles. The MIS2 protein is localized primarily on the plasma membrane along with the endosome. However, the Arg258Gln mutation located in extracellular domain in the mis2 mutant disturbed its subcellular localization. Additionally, three major haplotypes of MIS2 were identified in the japonica, indica and aus rice cultivars. The 18-bp InDel (insertion and deletion) in the 5'-UTR (untranslated region) caused different expression level of MIS2 in haplotypes. CONCLUSIONS: We reported a key role of OsCR4 in controlling grain size and shape by coordinately regulating epidermal cell size and cell number. The Arg258 in the extracellular seven-repeat domain is essential for the correct subcellular behavior and function of the OsCR4 protein.

Review of paediatric patients with urolithiasis, in view of development of urinary tract infection.

OBJECTIVE: To assess the development of UTI in paediatric patients, presented to OPD with urolithiasis. To ascertain what general parameters are associated with UTI, and examine specific characteristics of the calculi. METHOD: It was a retrospective study. Files of paediatric patients from July 2000 to December 2004 were reviewed. Only those patients with calculi and absent UTI and upto 5 years age were studied. All files of patients, primarily presenting with UTI, and those with documented urological procedures prior to UTI occurrence, were excluded from the study. Ultrasound and X-ray techniques were used to determine stone size and location. Collected urine samples were screened for UTI; organisms were isolated and cultured using Cystine Lactose Electrolyte Deficient (CLED) culture medium. RESULT: A total of 149 patients were studied. The mean age was 3.05 +/- 1.25 years, [77.2 %] were males [22.8%] females. Urinary tract infection [UTI] was found in 37.6% cases. Age status was significantly associated with UTI [p=0.008] along with the anatomical location [p=0.021]. The most common organism found on our culture plate of UTI positive patients was E. coli (21.4%). Bacteria were most sensitive to aminoglycoside group [16%] of antibiotics. CONCLUSION: We found a significant association between age, anatomical location of stones and UTI. These factors should be considered in paediatric patients to prevent UTI and its complications.