Comparative transcriptome profiling to unravel the key molecular signalling pathways and drought adaptive plasticity in shoot borne root system of sugarcane.

Sugarcane root system comprises of superficial sett roots as well as deeply-penetrating shoot borne roots (SBR) with latter being the permanent root system. In sugarcane, the healthy SBR contributes to a better crop yield and it also helps to produce multiple ratoon crops after the harvest. There is a dearth of in-depth knowledge on SBR system architecture and its functional role in modern day commercial hybrids. A comprehensive phenotypic, anatomical and whole transcriptome profiling, conducted between the commercial sugarcane hybrids and a wild germplasm Erianthus, found a developmental delay in both initiation and establishment of the SBR in commercial hybrid compared to Erianthus. The SBR system in Erianthus proved to be an extensive drought-adaptive root system architecture that significantly contributes to drought tolerance. On the other hand, SBRs in the commercial hybrids showed an irreversible collapse and damage of the root cells under drought stress. The outcomes from the comparative analysis of the transcriptome data showed a significant upregulation of the genes that regulate important stress signalling pathways viz., sugar, calcium, hormone signalling and phenylpropanoid biosynthesis in the SBRs of Erianthus. It was found that through these key signalling pathways, Erianthus SBRs triggered the downstream signalling cascade to impart physiological responses like osmoprotection, modification of the cell walls, detoxification of reactive oxygen species, expression of drought responsive transcription factors, maintenance of cell stability and lateral root development. The current study forms a basis for further exploration of the Shoot Borne Root system as a valuable breeding target to develop drought tolerant sugarcane genotypes.

Transgenic sugarcane with higher levels of BRK1 showed improved drought tolerance.

KEY MESSAGE: Transgenic sugarcane overexpressing BRK1 showed improved tolerance to drought stress through modulation of actin polymerization and formation of interlocking marginal lobes in epidermal leaf cells, a typical feature associated with BRK1 expression under drought stress. BRICK1 (BRK1) genes promote leaf epidermal cell morphogenesis and division in plants that involves local actin polymerization. Although the changes in actin filament organization during drought have been reported, the role of BRK in stress tolerance remains unknown. In our previous work, the drought-tolerant Erianthus arundinaceus exhibited high levels of the BRK gene expression under drought stress. Therefore, in the present study, the drought-responsive gene, BRK1 from Saccharum spontaneum, was transformed into sugarcane to test if it conferred drought tolerance in the commercial sugarcane cultivar Co 86032. The transgenic lines were subjected to drought stress, and analyzed using physiological parameters for drought stress. The drought-induced BRK1-overexpressing lines of sugarcane exhibited significantly higher transgene expression compared with the wild-type control and also showed improved physiological parameters. In addition, the formation of interlocking marginal lobes in the epidermal leaf cells, a typical feature associated with BRK1 expression, was observed in all transgenic BRK1 lines during drought stress. This is the first report to suggest that BRK1 plays a role in sugarcane acclimation to drought stress and may prove to be a potential candidate in genetic engineering of plants for enhanced biomass production under drought stress conditions.

Advances in Crop Breeding Through Precision Genome Editing.

The global climate change and unfavourable abiotic and biotic factors are limiting agricultural productivity and therefore intensifying the challenges for crop scientists to meet the rising demand for global food supply. The introduction of applied genetics to agriculture through plant breeding facilitated the development of hybrid varieties with improved crop productivity. However, the development of new varieties with the existing gene pools poses a challenge for crop breeders. Genetic engineering holds the potential to broaden genetic diversity by the introduction of new genes into crops. But the random insertion of foreign DNA into the plant's nuclear genome often leads to transgene silencing. Recent advances in the field of plant breeding include the development of a new breeding technique called genome editing. Genome editing technologies have emerged as powerful tools to precisely modify the crop genomes at specific sites in the genome, which has been the longstanding goal of plant breeders. The precise modification of the target genome, the absence of foreign DNA in the genome-edited plants, and the faster and cheaper method of genome modification are the remarkable features of the genome-editing technology that have resulted in its widespread application in crop breeding in less than a decade. This review focuses on the advances in crop breeding through precision genome editing. This review includes: an overview of the different breeding approaches for crop improvement; genome editing tools and their mechanism of action and application of the most widely used genome editing technology, CRISPR/Cas9, for crop improvement especially for agronomic traits such as disease resistance, abiotic stress tolerance, herbicide tolerance, yield and quality improvement, reduction of anti-nutrients, and improved shelf life; and an update on the regulatory approval of the genome-edited crops. This review also throws a light on development of high-yielding climate-resilient crops through precision genome editing.

Comparative de novo transcriptome analysis identifies salinity stress responsive genes and metabolic pathways in sugarcane and its wild relative Erianthus arundinaceus [Retzius] Jeswiet.

Erianthus arundinaceus [Retzius] Jeswiet, a wild relative of sugarcane has a high biomass production potential and a reservoir of many genes for superior agronomic traits and tolerance to biotic and abiotic stresses. A comparative physiological, anatomical and root transcriptome analysis were carried out to identify the salt-responsive genes and metabolic pathways associated with salt-tolerant E. arundinaceus genotype IND99-907 and salinity-sensitive sugarcane genotype Co 97010. IND99-907 recorded growth of young leaves, higher proline content, higher relative water content, intact root anatomical structures and lower Na+/K+, Ca2+/K+ and Mg2+/K+ ratio as compared to the sugarcane genotype Co 97010. We have generated four de novo transcriptome assemblies between stressed and control root samples of IND99-907 and Co 97010. A total of 649 and 501 differentially expressed genes (FDR<0.01) were identified from the stressed and control libraries of IND99-907 and Co 97010 respectively. Genes and pathways related to early stress-responsive signal transduction, hormone signalling, cytoskeleton organization, cellular membrane stabilization, plasma membrane-bound calcium and proton transport, sodium extrusion, secondary metabolite biosynthesis, cellular transporters related to plasma membrane-bound trafficking, nucleobase transporter, clathrin-mediated endocytosis were highly enriched in IND99-907. Whereas in Co 97010, genes related to late stress-responsive signal transduction, electron transport system, senescence, protein degradation and programmed cell death, transport-related genes associated with cellular respiration and mitochondrial respiratory chain, vesicular trafficking, nitrate transporter and fewer secondary metabolite biosynthetic genes were highly enriched. A total of 27 pathways, 24 biological processes, three molecular functions and one cellular component were significantly enriched (FDR≤ 0.05) in IND99-907 as compared to 20 pathways, two biological processes without any significant molecular function and cellular components in Co 97010, indicates the unique and distinct expression pattern of genes and metabolic pathways in both genotypes. The genomic resources developed from this study is useful for sugarcane crop improvement through development of genic SSR markers and genetic engineering approaches.

Isolation and characterization of water-deficit stress-responsive α-expansin 1 (EXPA1) gene from Saccharum complex.

In this study, full-length (1282-1330 bp) α-expansin 1 (EXPA1) gene from three different accessions belonging to Saccharum complex (Saccharum officinarum-SoEXPA1, Erianthus arundinaceus-EaEXPA1, and Saccharum spp. hybrid-ShEXPA1) was isolated using RAGE technique and characterized. The intronic and coding regions of isolated expansin genes ranged between 526-568 and 756-762 bp, respectively. An open reading frame encoding a polypeptide of 252 amino acids was obtained from S. officinarum and commercial sugarcane hybrid, whereas 254 amino acids were obtained in E. arundinaceus, a wild relative of Saccharum. Bioinformatics analysis of deduced protein revealed the presence of specific signature sequences and conserved amino acid residues crucial for the functioning of the protein. The predicted physicochemical characterization showed that the protein is stable in nature with instability index (II) value less than 40 and also clearly shown the dominance of random coil in the protein structure. Phylogenetic analysis revealed high conservation of EXPA1 among Saccharum complex and related crop species, Sorghum bicolor and Zea mays. The docking study of EXPA1 protein showed the interaction with xylose, which is present in xyloglucan of plant cell wall, elucidated the role of the expansin proteins in plant cell wall modification. This was further supported by the subcellular localization experiment in which it is clearly seen that the expansin protein localizes in the cell wall. Relative expression analysis of EXPA1 gene in Saccharum complex during drought stress showed high expression of the EaEXPA1 in comparison with SoEXPA1 and ShEXPA1 indicating possible role of EaEXPA1 in increased water-deficit stress tolerance in E. arundinaceus. These results suggest the potential use of EXPA1 for increasing the water-deficient stress tolerance levels in crop plants.

In silico characterisation and functional validation of chilling tolerant divergence 1 (COLD1) gene in monocots during abiotic stress.

The G protein-coupled receptor is one of the major transmembrane proteins in plants. It consists of an α subunit, a ß subunit and three γ subunits. Chilling tolerant divergence 1 (COLD1) includes a Golgi pH receptor (GPHR) domain, which maintains cell membrane organisation and dynamics, along with abscisic acid linked G protein-coupled receptor (ABA_GPCR) that regulates the signalling pathways during cold stress. In the present study, we performed characterisation of a homologous COLD1 from the economically important monocot species Oryza sativa L., Zea mays L., Sorghum bicolor (L.)Moench and Erianthus arundinaceus (L.) Beauv. IK 76-81, a wild relative of Saccharum. COLD1 was isolated from E. arundinaceus IK 76-81, analysed for its evolution, domain, membrane topology, followed by prediction of secondary, tertiary structures and functionally validated in all four different monocots. Gene expression studies of COLD1 revealed differential expression under heat, drought, salinity and cold stresses in selected monocots. This is the first study on regulation of native COLD1 during abiotic stress in monocots, which has opened up new leads for trait improvement strategies in this economically important crop species.

Corrigendum to: In silico characterisation and functional validation of chilling tolerant divergence 1 (COLD1) gene in monocots during abiotic stress.

The G protein-coupled receptor is one of the major transmembrane proteins in plants. It consists of an α subunit, a ß subunit and three γ subunits. Chilling tolerant divergence 1 (COLD1) includes a Golgi pH receptor (GPHR) domain, which maintains cell membrane organisation and dynamics, along with abscisic acid linked G protein-coupled receptor (ABA_GPCR) that regulates the signalling pathways during cold stress. In the present study, we performed characterisation of a homologous COLD1 from the economically important monocot species Oryza sativa L., Zea mays L., Sorghum bicolor (L.)Moench and Erianthus arundinaceus (L.) Beauv. IK 76-81, a wild relative of Saccharum. COLD1 was isolated from E. arundinaceus IK 76-81, analysed for its evolution, domain, membrane topology, followed by prediction of secondary, tertiary structures and functionally validated in all four different monocots. Gene expression studies of COLD1 revealed differential expression under heat, drought, salinity and cold stresses in selected monocots. This is the first study on regulation of native COLD1 during abiotic stress in monocots, which has opened up new leads for trait improvement strategies in this economically important crop species.

De novo sequencing and transcriptome analysis of a low temperature tolerant Saccharum spontaneum clone IND 00-1037.

Saccharum spontaneum L., a wild relative of sugarcane, is known for its adaptability to environmental stresses, particularly cold stress. In the present study, an attempt was made for transcriptome profiling of the low temperature (10°C) tolerant S. spontaneum clone IND 00-1037 collected from high altitude regions of Arunachal Pradesh, North Eastern India. The Illumina Nextseq500 platform yielded a total of 47.63 and 48.18 million reads corresponding to 4.7 and 4.8 gigabase pairs (Gb) of processed reads for control and cold stressed (10°C for 24h) samples, respectively. These reads were de novo assembled into 214,611 unigenes with an average length of 801bp. Further, all unigenes were aligned to GO, KEGG and COG databases in order to identify novel genes and pathways responsive upon low temperature conditions. The differential gene expression analysis revealed that about 2583 genes were upregulated and 3302 genes were down regulated during the stress. This is perhaps the comprehensive transcriptome data of a low temperature tolerant clone of S. spontaneum. This study would aid in identifying novel genes and also in future genomic studies pertaining to sugarcane and its wild relatives.

Introduction of Pea DNA Helicase 45 Into Sugarcane (Saccharum spp. Hybrid) Enhances Cell Membrane Thermostability And Upregulation Of Stress-responsive Genes Leads To Abiotic Stress Tolerance.

DNA helicases are motor proteins that play an essential role in nucleic acid metabolism, by providing a duplex-unwinding function. To improve the drought and salinity tolerance of sugarcane, a DEAD-box helicase gene isolated from pea with a constitutive promoter, Port Ubi 2.3 was transformed into the commercial sugarcane variety Co 86032 through Agrobacterium-mediated transformation, and the transgenics were screened for tolerance to soil moisture stress and salinity. The transgene integration was confirmed through polymerase chain reaction, and the V 0 transgenic events showed significantly higher cell membrane thermostability under normal irrigated conditions. The V 1 transgenic events were screened for tolerance to soil moisture stress and exhibited significantly higher cell membrane thermostability, transgene expression, relative water content, gas exchange parameters, chlorophyll content, and photosynthetic efficiency under soil moisture stress compared to wild-type (WT). The overexpression of PDH45 transgenic sugarcane also led to the upregulation of DREB2-induced downstream stress-related genes. The transgenic events demonstrated higher germination ability and better chlorophyll retention than WT under salinity stress. Our results suggest the possibility for development of increased abiotic stress tolerant sugarcane cultivars through overexpression of PDH45 gene. Perhaps this is the first report, which provides evidence for increased drought and salinity tolerance in sugarcane through overexpression of PDH45.

Erianthus arundinaceus HSP70 (EaHSP70) overexpression increases drought and salinity tolerance in sugarcane (Saccharum spp. hybrid).

Heat shock proteins (HSPs) have a major role in stress tolerance mechanisms in plants. Our studies have shown that the expression of HSP70 is enhanced under water stress in Erianthus arundinaceus. In this paper, we evaluate the effects of overexpression of EaHSP70 driven by Port Ubi 2.3 promoter in sugarcane. The transgenic events exhibit significantly higher gene expression, cell membrane thermostability, relative water content, gas exchange parameters, chlorophyll content and photosynthetic efficiency. The overexpression of EaHSP70 transgenic sugarcane led to the upregulation of stress-related genes. The transformed sugarcane plants had better chlorophyll retention and higher germination ability than control plants under salinity stress. Our results suggest that EaHSP70 plays an important role in sugarcane acclimation to drought and salinity stresses and its potential for genetic engineering of sugarcane for drought and salt tolerance.

Overexpression of EaDREB2 and pyramiding of EaDREB2 with the pea DNA helicase gene (PDH45) enhance drought and salinity tolerance in sugarcane (Saccharum spp. hybrid).

KEY MESSAGE: EaDREB2 overexpressed in sugarcane enhanced tolerance to drought and salinity. When co-transformed with plant DNA helicase gene, DREB2 showed greater level of salinity tolerance than in single-gene transgenics. Drought is one of the most challenging agricultural issues limiting sustainable sugarcane production and can potentially cause up to 50 % yield loss. DREB proteins play a vital regulatory role in abiotic stress tolerance in plants. We previously reported that expression of EaDREB2 is enhanced by drought stress in Erianthus arundinaceus. In this study, we have isolated the DREB2 gene from E. arundinaceus, transformed one of the most popular sugarcane variety Co 86032 in tropical India with EaDREB2 through Agrobacterium-mediated transformation, pyramided the EaDREB2 gene with the gene coding for PDH45 driven by Port Ubi 2.3 promoter through particle bombardment and evaluated the V1 transgenics for soil deficit moisture and salinity stresses. Soil moisture stress was imposed at the tillering phase by withholding irrigation. Physiological, molecular and morphological parameters were used to assess drought tolerance. Salinity tolerance was assessed through leaf disc senescence and bud sprout assays under salinity stress. Our results indicate that overexpression of EaDREB2 in sugarcane enhances drought and salinity tolerance to a greater extent than the untransformed control plants. This is the first report of the co-transformation of EaDREB2 and PDH45 which shows higher salinity tolerance but lower drought tolerance than EaDREB2 alone. The present study seems to suggest that, for combining drought and salinity tolerance together, co-transformation is a better approach.

Isolation and symbiotic characteristics of two Tn5-derived phage-resistant Bradyrhizobium japonicum strains that nodulate soybean.

Using transponson Tn5 mutagenesis, two transconjugants of Bradyrhizobium japonicum with the properties of both phage resistance and ability to induce nodulation were isolated at the frequency of 0.02%. These transconjugants were tested for their symbiotic performance on soybean cv. JS335 under greenhouse and field conditions. Both phage-resistant mutants induced nodules (nod (+)), but the transconjugant B. japonicum E13 was ineffective in nitrogen fixation (fix (-)). Rhizobiophage presence in the inoculum of phage-resistant mutants did not influence the symbiotic effectiveness. The mixture of wild strain and phage in the inoculum caused reduced symbiotic performance under controlled conditions, while under a field environment phage (100 and 500 mul of approximately 10(8) particles ml(-1)) presence did not have any recognizable effect on increased nodule dry weight, nitrogenase activity, or foliar N(2) content. On the basis of restriction fragment length polymorphism analysis, phage-sensitive, less effective, homologous bradyrhizobia belonging to B. japonicum were detected in root nodules of both inoculated and uninoculated plants. Inoculation of a higher concentration of phage in the inoculum significantly reduced the symbiotic performance, while the lower concentration of phage did not show any effect on phage-susceptible, less effective, homologous bradyrhizobia or, thus, symbiotic efficiency under field conditions. The phage-resistant mutant B. japonicum A49 showed effective symbiosis as efficient as that of the wild strain. Inoculation of phage-resistant mutants with lytic phage may reduce the occupancy of phage-susceptible, ineffective/less effective/mediocre homologous bradyrhizobia strains under natural complex soil conditions.

Regulation of urease in Bradyrhizobium colonizing green gram (Vigna radiata (L.) Wilczek).

In the present study attempts have been made to characterize urease expression in slow growing Bradyrhizobium strains TAL442 and MO5 which are endosymbionts of green gram (Vigna radiata (L.)Wilczek). It was found that urease activity in vegetative cells of both the strains was inducible unlike their fast growing counterparts. Mode of regulation in TAL442 was governed by presence of ammonia. Urease expression was also detectable in bacteroids of both the strains which was not influenced by presence of external nickel chloride in high concentration, a situation detrimental to the vegetative cells.

Morphology and general characteristics of lytic phages infective on strains of Bradyrhizobium japonicum.

Biological characteristics of three isolated phages (SR1, SR2, and SR3) lytic against three Bradyrhizobium japonicum strains were studied. These phages had no cross-infectivity among the host strains. Phage morphology indicates that they belonged to Siphoviridae (long noncontractile tail; SR1 and SR2) and Podoviridae (short tail; SR3) classes of bacteriophages. Lytic cycle of phages studied under identical conditions showed a distinct adsorption rate (67.3-99.1%), latent period (150-300 min), rise period (60-150 min), and burst size (110-200 pfu/cell). Stability in liquids and inactivation by osmotic shock, thermal, and ultraviolet irradiation were also distinct in this heterogeneous phage group. Influence of soil factors such as temperature, soil moisture, soil pH, and degree of phage adsorption to the soil on phage survival was determined. Major percent of free infective phages were obtained after desorption of phages from soil. Overall, temperature appeared to be the most important parameter affecting rhizobiophage survival in the soil.

Phage typing of indigenous soybean-rhizobia and relationship of a phage group strains for their asymbiotic and symbiotic nitrogen fixation.

A total of 354 indigenous bradyrhizobia were isolated from soybean nodules collected from five major crop grown regions. Host-specific 12 phages, each active on particular strains were selected. Factors, which influence the interaction between the host and phage, were examined. Four different types of plaques were detected. Nearly 17% of isolates were found resistant to all phages. Phage sensitivity patterns revealed a total of 32 distinct phage genotype groups. Different set of phage combinations expressed variation in specificity for parasitizing against particular group of rhizobia. Distributions of isolates in each phage types differed markedly between regions. Interestingly, nine strains belonging to phage group 16 exhibited high ex planta nitrogenase activity in culture. However, no correlation could be established between high ex planta nitrogenase activity and their symbiotic effectiveness with soybean cultivars. Soybean cv. JS335 showed relatively superior performance than Bragg and Lee with indigenous bradyrhizobial strains. Phage typing revealed the existence of large genetic diversity among native rhizobia and selection of the superior bradyrhizobial strains can also be possible for a given soil-climate-cultivar complex.