Introgression of Δ1-pyrroline-5-carboxylate synthetase (PgP5CS) confers enhanced resistance to abiotic stresses in transgenic tobacco.

Δ1-pyrroline-5-carboxylate synthetase (P5CS) is one of the key regulatory enzymes involved in the proline biosynthetic pathway. Proline acts as an osmoprotectant, molecular chaperone, antioxidant, and regulator of redox homeostasis. The accumulation of proline during stress is believed to confer tolerance in plants. In this study, we cloned the complete CDS of the P5CS from pearl millet (Pennisetum glaucum (L.) R.Br. and transformed into tobacco. Three transgenic tobacco plants with single-copy insertion were analyzed for drought and heat stress tolerance. No difference was observed between transgenic and wild-type (WT) plants when both were grown in normal conditions. However, under heat and drought, transgenic plants have been found to have higher chlorophyll, relative water, and proline content, and lower malondialdehyde (MDA) levels than WT plants. The photosynthetic parameters (stomatal conductance, intracellular CO2 concentration, and transpiration rate) were also observed to be high in transgenic plants under abiotic stress conditions. qRT-PCR analysis revealed that the expression of the transgene in drought and heat conditions was 2-10 and 2-7.5 fold higher than in normal conditions, respectively. Surprisingly, only P5CS was increased under heat stress conditions, indicating the possibility of feedback inhibition. Our results demonstrate the positive role of PgP5CS in enhancing abiotic stress tolerance in tobacco, suggesting its possible use to increase abiotic stress-tolerance in crops for sustained yield under adverse climatic conditions.

An Optimized and Cost-Effective RNA Extraction Method for Secondary Metabolite-Enriched Tissues of Norway Spruce (Picea abies).

Since the development of next-generation sequencing techniques and with the growing interest in transcriptomic studies, there is a demand for high-throughput RNA extraction techniques. General RNA extraction protocols are unreliable when it comes to the quality and quantity of isolated RNA obtained from different tissue types of different plant species. Despite Norway spruce (Picea abies) being one of the most significant and commercially valuable tree species in European forests, only limited genetic research is available. In this study, we developed a cetyltrimethylammonium bromide (CTAB) protocol by modifying the original method. We compared this CTAB protocol with other widely used methods for extracting RNA from different tissues (needle, phloem, and root) of Norway spruce, known for its richness in polyphenols, polysaccharides, and secondary metabolites. The modified CTAB method proves to be superior to the kit-based and TRIzol-based methods for extracting RNA from the metabolite-rich tissues of Norway spruce, resulting in high RNA quality and integrity values (RIN~7-9). The modified CTAB RNA extraction method is rapid, cost-effective, and relatively simple in yielding the desired RNA quality from Norway spruce tissues. It is optimal for RNA sequencing and other downstream molecular applications.

Gene expression plasticity facilitates different host feeding in Ips sexdentatus (Coleoptera: Curculionidae: Scolytinae).

Host shift is ecologically advantageous and a crucial driver for herbivore insect speciation. Insects on the non-native host obtain enemy-free space and confront reduced competition, but they must adapt to survive. Such signatures of adaptations can often be detected at the gene expression level. It is astonishing how bark beetles cope with distinct chemical environments while feeding on various conifers. Hence, we aim to disentangle the six-toothed bark beetle (Ips sexdentatus) response against two different conifer defences upon host shift (Scots pine to Norway spruce). We conducted bioassay and metabolomic analysis followed by RNA-seq experiments to comprehend the beetle's ability to surpass two different terpene-based conifer defence systems. Beetle growth rate and fecundity were increased when reared exclusively on spruce logs (alternative host) compared to pine logs (native host). Comparative gene expression analysis identified differentially expressed genes (DEGs) related to digestion, detoxification, transporter activity, growth, signalling, and stress response in the spruce-feeding beetle gut. Transporter genes were highly abundant during spruce feeding, suggesting they could play a role in pumping a wide variety of endogenous and xenobiotic compounds or allelochemicals out. Trehalose transporter (TRET) is also up-regulated in the spruce-fed beetle gut to maintain homeostasis and stress tolerance. RT-qPCR and enzymatic assays further corroborated some of our findings. Taken together, the transcriptional plasticity of key physiological genes plays a crucial role after the host shift and provides vital clues for the adaptive potential of bark beetles on different conifer hosts.

Comparative gut proteomics study revealing adaptive physiology of Eurasian spruce bark beetle, Ips typographus (Coleoptera: Scolytinae).

The bark beetle, Ips typographus (L.), is a major pest of Norway spruce, Picea abies (L.), causing enormous economic losses globally. The adult stage of the I. typographus has a complex life cycle (callow and sclerotized); the callow beetles feed ferociously, whereas sclerotized male beetles are more aggressive and pioneers in establishing new colonies. We conducted a comparative proteomics study to understand male and female digestion and detoxification processes in callow and sclerotized beetles. Proteome profiling was performed using high-throughput liquid chromatography-mass spectrometry. A total of >3000 proteins were identified from the bark beetle gut, and among them, 539 were differentially abundant (fold change ±2, FDR <0.05) between callow and sclerotized beetles. The differentially abundant proteins (DAPs) mainly engage with binding, catalytic activity, anatomical activity, hydrolase activity, metabolic process, and carbohydrate metabolism, and hence may be crucial for growth, digestion, detoxification, and signalling. We validated selected DAPs with RT-qPCR. Gut enzymes such as NADPH-cytochrome P450 reductase (CYC), glutathione S-transferase (GST), and esterase (EST) play a crucial role in the I. typographus for detoxification and digesting of host allelochemicals. We conducted enzyme activity assays with them and observed a positive correlation of CYC and GST activities with the proteomic results, whereas EST activity was not fully correlated. Furthermore, our investigation revealed that callow beetles had an upregulation of proteins associated with juvenile hormone (JH) biosynthesis and chitin metabolism, whereas sclerotized beetles exhibited an upregulation of proteins linked to fatty acid metabolism and the TCA cycle. These distinctive patterns of protein regulation in metabolic and functional processes are specific to each developmental stage, underscoring the adaptive responses of I. typographicus in overcoming conifer defences and facilitating their survival. Taken together, it is the first gut proteomic study comparing males and females of callow and sclerotized I. typographus, shedding light on the adaptive ecology at the molecular level. Furthermore, the information about bark beetle handling of nutritionally limiting and defence-rich spruce phloem diet can be utilized to formulate RNAi-mediated beetle management.

A conserved SNP variation in the pre-miR396c flanking region in Oryza sativa indica landraces correlates with mature miRNA abundance.

Plant precursor miRNAs (pre-miRNA) have conserved evolutionary footprints that correlate with mode of miRNA biogenesis. In plants, base to loop and loop to base modes of biogenesis have been reported. Conserved structural element(s) in pre-miRNA play a major role in turn over and abundance of mature miRNA. Pre-miR396c sequences and secondary structural characteristics across Oryza species are presented. Based on secondary structure, twelve Oryza pre-miR396c sequences are divided into three groups, with the precursor from halophytic Oryza coarctata forming a distinct group. The miRNA-miRNA* duplex region is completely conserved across eleven Oryza species as are other structural elements in the pre-miRNA, suggestive of an evolutionarily conserved base-to-loop mode of miRNA biogenesis. SNPs within O. coarctata mature miR396c sequence and miRNA* region have the potential to alter target specificity and association with the RNA-induced silencing complex. A conserved SNP variation, rs10234287911 (G/A), identified in O. sativa pre-miR396c sequences alters base pairing above the miRNA-miRNA* duplex. The more stable structure conferred by the 'A10234287911' allele may promote better processing vis-à-vis the structure conferred by 'G10234287911' allele. We also examine pri- and pre-miR396c expression in cultivated rice under heat and salinity and their correlation with miR396c expression.

Comparative Analysis of Root Na+ Relation under Salinity between Oryza sativa and Oryza coarctata

Na+ toxicity is one of the major physiological constraints imposed by salinity on plant performance. At the same time, Na+ uptake may be beneficial under some circumstances as an easily accessible inorganic ion that can be used for increasing solute concentrations and maintaining cell turgor. Two rice species, Oryza sativa (cultivated rice, salt-sensitive) and Oryza coarctata (wild rice, salt-tolerant), demonstrated different strategies in controlling Na+ uptake. Glasshouse experiments and gene expression analysis suggested that salt-treated wild rice quickly increased xylem Na+ loading for osmotic adjustment but maintained a non-toxic level of stable shoot Na+ concentration by increased activity of a high affinity K+ transporter HKT1;5 (essential for xylem Na+ unloading) and a Na+/H+ exchanger NHX (for sequestering Na+ and K+ into root vacuoles). Cultivated rice prevented Na+ uptake and transport to the shoot at the beginning of salt treatment but failed to maintain it in the long term. While electrophysiological assays revealed greater net Na+ uptake upon salt application in cultivated rice, O. sativa plants showed much stronger activation of the root plasma membrane Na+/H+ Salt Overly Sensitive 1 (SOS1) exchanger. Thus, it appears that wild rice limits passive Na+ entry into root cells while cultivated rice relies heavily on SOS1-mediating Na+ exclusion, with major penalties imposed by the existence of the "futile cycle" at the plasma membrane.

Identifying optimal reference genes for gene expression studies in Eurasian spruce bark beetle, Ips typographus (Coleoptera: Curculionidae: Scolytinae).

Eurasian spruce bark beetle (Ips typographus [L.]) causes substantial damage to spruce forests worldwide. Undoubtedly, more aggressive measures are necessary to restrict the enduring loss. Finishing genome sequencing is a landmark achievement for deploying molecular techniques (i.e., RNA interference) to manage this pest. Gene expression studies assist in understanding insect physiology and deployment of molecular approaches for pest management. RT-qPCR is a valuable technique for such studies. However, accuracy and reliability depend on suitable reference genes. With the genome sequence available and the growing requirement of molecular tools for aggressive forest pest management, it is crucial to find suitable reference genes in Ips typographus under different experimental conditions. Hence, we evaluated the stability of twelve candidate reference genes under diverse experimental conditions such as biotic (developmental, sex and tissues) and abiotic factors (i.e., temperature and juvenile hormone treatment) to identify the reference genes. Our results revealed that ribosomal protein 3a (RPS3-a) was the best reference gene across all the experimental conditions, with minor exceptions. However, the stability of the reference gene can differ based on experiments. Nevertheless, present study provides a comprehensive list of reference genes under different experimental conditions for Ips typographus and contributes to "future genomic and functional genomic research".

Unravelling the physiological basis of salinity stress tolerance in cultivated and wild rice species.

Wild rice species provide a rich source of genetic diversity for possible introgression of salinity stress tolerance in cultivated rice. We investigated the physiological basis of salinity stress tolerance in Oryza species by using six rice genotypes (Oryza sativa L.) and four wild rice species. Three weeks of salinity treatment significantly (P <0.05) reduced physiological and growth indices of all cultivated and wild rice lines. However, the impact of salinity-induced growth reduction differed substantially among accessions. Salt tolerant accessions showed better control over gas exchange properties, exhibited higher tissue tolerance, and retained higher potassium ion content despite higher sodium ion accumulation in leaves. Wild rice species showed relatively lower and steadier xylem sap sodium ion content over the period of 3weeks analysed, suggesting better control over ionic sodium xylem loading and its delivery to shoots with efficient vacuolar sodium ion sequestration. Contrary to this, saline sensitive genotypes managed to avoid initial Na+ loading but failed to accomplish this in the long term and showed higher sap sodium ion content. Conclusively, our results suggest that wild rice genotypes have more efficient control over xylem sodium ion loading, rely on tissue tolerance mechanisms and allow for a rapid osmotic adjustment by using sodium ions as cheap osmoticum for osmoregulation.

Proto Kranz-like leaf traits and cellular ionic regulation are associated with salinity tolerance in a halophytic wild rice.

Species of wild rice (Oryza spp.) possess a wide range of stress tolerance traits that can be potentially utilized in breeding climate-resilient cultivated rice cultivars (Oryza sativa) thereby aiding global food security. In this study, we conducted a greenhouse trial to evaluate the salinity tolerance of six wild rice species, one cultivated rice cultivar (IR64) and one landrace (Pokkali) using a range of electrophysiological, imaging, and whole-plant physiological techniques. Three wild species (O. latifolia, O. officinalis and O. coarctata) were found to possess superior salinity stress tolerance. The underlying mechanisms, however, were strikingly different. Na+ accumulation in leaves of O. latifolia, O. officinalis and O. coarctata were significantly higher than the tolerant landrace, Pokkali. Na+ accumulation in mesophyll cells was only observed in O. coarctata, suggesting that O. officinalis and O. latifolia avoid Na+ accumulation in mesophyll by allocating Na+ to other parts of the leaf. The finding also suggests that O. coarctata might be able to employ Na+ as osmolyte without affecting its growth. Further study of Na+ allocation in leaves will be helpful to understand the mechanisms of Na+ accumulation in these species. In addition, O. coarctata showed Proto Kranz-like leaf anatomy (enlarged bundle sheath cells and lower numbers of mesophyll cells), and higher expression of C4-related genes (e.g., NADPME, PPDK) and was a clear outlier with respect to salinity tolerance among the studied wild and cultivated Oryza species. The unique phylogenetic relationship of O. coarctata with C4 grasses suggests the potential of this species for breeding rice with high photosynthetic rate under salinity stress in the future.

Reduced apoplastic barriers in tissues of shoot-proximal rhizomes of Oryza coarctata are associated with Na+ sequestration.

Oryza coarctata is the only wild rice species with significant salinity tolerance. The present work examines the role of the substantial rhizomatous tissues of O. coarctata in conferring salinity tolerance. Transition to an erect phenotype (shoot emergence) from prostrate growth of rhizome tissues is characterized by marked lignification and suberization of supporting sclerenchymatous tissue, epidermis, and bundle sheath cells in aerial shoot-proximal nodes and internodes in O. coarctata. With salinity, however, aerial shoot-proximal internodal tissues show reductions in lignification and suberization, most probably related to re-direction of carbon flux towards synthesis of the osmporotectant proline. Concurrent with hypolignification and reduced suberization, the aerial rhizomatous biomass of O. coarctata appears to have evolved mechanisms to store Na+ in these specific tissues under salinity. This was confirmed by histochemical staining, quantitative real-time reverse transcription-PCR expression patterns of genes involved in lignification/suberization, Na+ and K+ contents of internodal tissues, as well as non-invasive microelectrode ion flux measurements of NaCl-induced net Na+, K+, and H+ flux profiles of aerial nodes were determined. In O. coarctata, aerial proximal internodes appear to act as 'traffic controllers', sending required amounts of Na+ and K+ into developing leaves for osmotic adjustment and turgor-driven growth, while more deeply positioned internodes assume a Na+ buffering/storage role.

Reference Gene Selection for Normalizing Gene Expression in Ips Sexdentatus (Coleoptera: Curculionidae: Scolytinae) Under Different Experimental Conditions.

Ips sexdentatus (Coleoptera: Curculionidae: Scolytinae) is one of the most destructive and economically important forest pests. A better understanding of molecular mechanisms underlying its adaptation to toxic host compounds may unleash the potential for future management of this pest. Gene expression studies could be considered as one of the key experimental approaches for such purposes. A suitable reference gene selection is fundamental for quantitative gene expression analysis and functional genomics studies in I. sexdentatus. Twelve commonly used reference genes in Coleopterans were screened under different experimental conditions to obtain accurate and reliable normalization of gene expression data. The majority of the 12 reference genes showed a relatively stable expression pattern among developmental stages, tissue-specific, and sex-specific stages; however, some variabilities were observed during varied temperature incubation. Under developmental conditions, the Tubulin beta-1 chain (ß-Tubulin) was the most stable reference gene, followed by translation elongation factor (eEF2) and ribosomal protein S3 (RPS3). In sex-specific conditions, RPS3, ß-Tubulin, and eEF2 were the most stable reference genes. In contrast, different sets of genes were shown higher stability in terms of expression under tissue-specific conditions, i.e., RPS3 and eEF2 in head tissue, V-ATPase-A and eEF2 in the fat body, V-ATPase-A and eEF2 in the gut. Under varied temperatures, ß-Tubulin and V-ATPase-A were most stable, whereas ubiquitin (UbiQ) and V-ATPase-A displayed the highest expression stability after Juvenile Hormone III treatment. The findings were validated further using real-time quantitative reverse transcription PCR (RT-qPCR)-based target gene expression analysis. Nevertheless, the present study delivers a catalog of reference genes under varied experimental conditions for the coleopteran forest pest I. sexdentatus and paves the way for future gene expression and functional genomic studies on this species.

To exclude or to accumulate? Revealing the role of the sodium HKT1;5 transporter in plant adaptive responses to varying soil salinity.

Arid/semi-arid and coastal agricultural areas of the world are especially vulnerable to climate change-driven soil salinity. Salinity tolerance in plants is a complex trait, with salinity negatively affecting crop yield. Plants adopt a range of mechanisms to combat salinity, with many transporter genes being implicated in Na+-partitioning processes. Within these, the high-affinity K+ (HKT) family of transporters play a critical role in K+ and Na+ homeostasis in plants. Among HKT transporters, Type I transporters are Na+-specific. While Arabidopsis has only one Na + -specific HKT (AtHKT1;1), cereal crops have a multiplicity of Type I and II HKT transporters. AtHKT1; 1 (Arabidopsis thaliana) and HKT1; 5 (cereal crops) 'exclude' Na+ from the xylem into xylem parenchyma in the root, reducing shoot Na+ and hence, confer sodium tolerance. However, more recent data from Arabidopsis and crop species show that AtHKT1;1/HKT1;5 alleles have a strong genetic association with 'shoot sodium accumulation' and concomitant salt tolerance. The review tries to resolve these two seemingly contradictory effects of AtHKT1;1/HKT1;5 operation (shoot exclusion vs shoot accumulation), both conferring salinity tolerance and suggests that contrasting phenotypes are attributable to either hyper-functional or weak AtHKT1;1/HKT1;5 alleles/haplotypes and are under strong selection by soil salinity levels. It also suggests that opposite balancing mechanisms involving xylem ion loading in these contrasting phenotypes exist that require transporters such as SOS1 and CCC. While HKT1; 5 is a crucial but not sole determinant of salinity tolerance, investigation of the adaptive benefit(s) conferred by naturally occurring intermediate HKT1;5 alleles will be important under a climate change scenario.

Distinct Evolutionary Origins of Intron Retention Splicing Events in NHX1 Antiporter Transcripts Relate to Sequence Specific Distinctions in Oryza Species.

The genome of Asian cultivated rice (Oryza sativa L.) shows the presence of six organelle-specific and one plasma membrane (OsNHX1-7) NHX-type cation proton antiporters. Of these, vacuolar-localized OsNHX1 is extensively characterized. The genus Oryza consists of 27 species and 11 genome-types, with cultivated rice, diploid O. sativa, having an AA-type genome. Oryza NHX1 orthologous regions (gene organization, 5' upstream cis elements, amino acid residues/motifs) from closely related Oryza AA genomes cluster distinctly from NHX1 regions from more ancestral Oryza BB, FF and KKLL genomes. These sequence-specific distinctions also extend to two separate intron retention (IR) events involving Oryza NHX1 transcripts that occur at the 5' and 3' ends of the NHX1 transcripts. We demonstrate that the IR event involving the 5' UTR is present only in more recently evolved Oryza AA genomes while the IR event governing retention of the 13th intron of Oryza NHX1 (terminal intron) is more ancient in origin, also occurring in halophytic wild rice, Oryza coarctata (KKLL). We also report presence of a retro-copy of the OcNHX1 cDNA in the genome of O. coarctata (rOcNHX1). Preferential species and tissue specific up- or down-regulation of the correctly spliced NHX1 transcript/5' UTR/13th intron-retaining splice variants under salinity was observed. The implications of IR on NHX1 mRNA stability and ORF diversity in Oryza spp. is discussed.

Microhair on the adaxial leaf surface of salt secreting halophytic Oryza coarctata Roxb. show distinct morphotypes: Isolation for molecular and functional analysis.

Halophytic Oryza coarctata is a good model system to examine mechanisms of salinity tolerance in rice. O. coarctata leaves show the presence of microhairs in adaxial leaf surface furrows that secrete salt under salinity. However, detailed molecular and physiological studies of O. coarctata microhairs are limited due to their relative inaccessibility. This work presents a detailed characterization of O. coarctata leaf features. O. coarctata has two types of microhairs on the adaxial leaf surface: longer microhairs (three morphotypes) lining epidermal furrow walls and shorter microhairs (reported first time) arising from bulliform cells. Microhair morphotypes include (i) finger-like, tubular structures, (ii) tubular hairs with bilobed and flattened heads and (iii) bi-or trifurcated hairs. The unicellular nature of microhairs was confirmed by propidium iodide (PI) staining. An efficient method for the isolation and enrichment of O. coarctata microhairs is presented (yield averaging ˜2 × 105/g leaf tissue). The robustness of the microhair isolation procedure was confirmed by subsequent viability staining (PI), total RNA isolation and RT-PCR amplification of O. coarctata trichome-specific WUSCHEL-related homeobox 3B (OcWox3B) and transporter gene-specific cDNA sequences. The present microhair isolation work from O. coarctata paves the way for examining genes involved in ion secretion in this halophytic wild rice model.

CRISPR for Crop Improvement: An Update Review.

The availability of genome sequences for several crops and advances in genome editing approaches has opened up possibilities to breed for almost any given desirable trait. Advancements in genome editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) has made it possible for molecular biologists to more precisely target any gene of interest. However, these methodologies are expensive and time-consuming as they involve complicated steps that require protein engineering. Unlike first-generation genome editing tools, CRISPR/Cas9 genome editing involves simple designing and cloning methods, with the same Cas9 being potentially available for use with different guide RNAs targeting multiple sites in the genome. After proof-of-concept demonstrations in crop plants involving the primary CRISPR-Cas9 module, several modified Cas9 cassettes have been utilized in crop plants for improving target specificity and reducing off-target cleavage (e.g., Nmcas9, Sacas9, and Stcas9). Further, the availability of Cas9 enzymes from additional bacterial species has made available options to enhance specificity and efficiency of gene editing methodologies. This review summarizes the options available to plant biotechnologists to bring about crop improvement using CRISPR/Cas9 based genome editing tools and also presents studies where CRISPR/Cas9 has been used for enhancing biotic and abiotic stress tolerance. Application of these techniques will result in the development of non-genetically modified (Non-GMO) crops with the desired trait that can contribute to increased yield potential under biotic and abiotic stress conditions.