Deciphering the differential expression patterns of yield-related negative regulators in hexaploid wheat cultivars and hybrids at different growth stages.

BACKGROUND: Hexaploid bread wheat underwent a series of polyploidization events through interspecific hybridizations that conferred adaptive plasticity and resulted in duplication and neofunctionalization of major agronomic genes. The genetic architecture of polyploid wheat not only confers adaptive plasticity but also offers huge genetic diversity. However, the contribution of different gene copies (homeologs) encoded from different subgenomes (A, B, D) at different growth stages remained unexplored. METHODS: In this study, hybrid of elite cultivars of wheat were developed via reciprocal crosses (cytoplasm swapping) and phenotypically evaluated. We assessed differential expression profiles of yield-related negative regulators in these cultivars and their F1 hybrids and identified various cis-regulatory signatures by employing bioinformatics tools. Furthermore, the preferential expression patterns of the syntenic triads encoded from A, B, and D subgenomes were assessed to decipher their functional redundancy at six different growth stages. RESULTS: Hybrid progenies showed better heterosis such as up to 17% increase in the average number of grains and up to 50% increase in average thousand grains weight as compared to mid-parents. Based on the expression profiling, our results indicated significant dynamic transcriptional expression patterns, portraying the different homeolog-dominance at the same stage in the different cultivars and their hybrids. Albeit belonging to same syntenic triads, a dynamic trend was observed in the regulatory signatures of these genes that might be influencing their expression profiles. CONCLUSION: These findings can substantially contribute and provide insights for the selective introduction of better cultivars into traditional and hybrid breeding programs which can be harnessed for the improvement of future wheat.

AtCIPK16, a CBL-interacting protein kinase gene, confers salinity tolerance in transgenic wheat.

Globally, wheat is the major source of staple food, protein, and basic calories for most of the human population. Strategies must be adopted for sustainable wheat crop production to fill the ever-increasing food demand. Salinity is one of the major abiotic stresses involved in plant growth retardation and grain yield reduction. In plants, calcineurin-B-like proteins form a complicated network with the target kinase CBL-interacting protein kinases (CIPKs) in response to intracellular calcium signaling as a consequence of abiotic stresses. The AtCIPK16 gene has been identified in Arabidopsis thaliana and found to be significantly upregulated under salinity stress. In this study, the AtCIPK16 gene was cloned in two different plant expression vectors, i.e., pTOOL37 having a UBI1 promoter and pMDC32 having a 2XCaMV35S constitutive promoter transformed through the Agrobacterium-mediated transformation protocol, in the local wheat cultivar Faisalabad-2008. Based on their ability to tolerate different levels of salt stress (0, 50, 100, and 200 mM), the transgenic wheat lines OE1, OE2, and OE3 expressing AtCIPK16 under the UBI1 promoter and OE5, OE6, and OE7 expressing the same gene under the 2XCaMV35S promoter performed better at 100 mM of salinity stress as compared with the wild type. The AtCIPK16 overexpressing transgenic wheat lines were further investigated for their K+ retention ability in root tissues by utilizing the microelectrode ion flux estimation technique. It has been demonstrated that after 10 min of 100 mM NaCl application, more K+ ions were retained in the AtCIPK16 overexpressing transgenic wheat lines than in the wild type. Moreover, it could be concluded that AtCIPK16 functions as a positive elicitor in sequestering Na+ ions into the cell vacuole and retaining more cellular K+ under salt stress to maintain ionic homeostasis.

32Pi Labeled Transgenic Wheat Shows the Accumulation of Phosphatidylinositol 4,5-bisphosphate and Phosphatidic Acid Under Heat and Osmotic Stress.

The ensuing heat stress drastically affects wheat plant growth and development, consequently compromising its grain yield. There are many thermoregulatory processes/mechanisms mediated by ion channels, lipids, and lipid-modifying enzymes that occur in the plasma membrane and the chloroplast. With the onset of abiotic or biotic stresses, phosphoinositide-specific phospholipase C (PI-PLC), as a signaling enzyme, hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which is further phosphorylated into phosphatidic acid (PA) as a secondary messenger and is involved in multiple processes. In the current study, a phospholipase C (PLC) signaling pathway was investigated in spring wheat (Triticum aestivum L.) and evaluated its four AtPLC5 overexpressed (OE)/transgenic lines under heat and osmotic stresses through 32Pi radioactive labeling. Naturally, the wheat harbors only a small amount of PIP2. However, with the sudden increase in temperature (40°C), PIP2 levels start to rise within 7.5 min in a time-dependent manner in wild-type (Wt) wheat. While the Phosphatidic acid (PA) level also elevated up to 1.6-fold upon exposing wild-type wheat to heat stress (40°C). However, at the anthesis stage, a significant increase of ∼4.5-folds in PIP2 level was observed within 30 min at 40°C in AtPLC5 over-expressed wheat lines. Significant differences in PIP2 level were observed in Wt and AtPLC5-OE lines when treated with 1200 mM sorbitol solution. It is assumed that the phenomenon might be a result of the activation of PLC/DGK pathways. Together, these results indicate that heat stress and osmotic stress activate several lipid responses in wild-type and transgenic wheat and can explain heat and osmotic stress tolerance in the wheat plant.

Genome edited wheat- current advances for the second green revolution.

Common wheat is a major source of nutrition around the globe, but unlike maize and rice hybrids, no breakthrough has been made to enhance wheat yield since Green Revolution. With the availability of reference genome sequence of wheat and advancement of allied genomics technologies, understanding of genes involved in grain yield components and disease resistance/susceptibility has opened new avenues for crop improvement. Wheat has a huge hexaploidy genome of approximately 17 GB with 85% repetition, and it is a daunting task to induce any mutation across three homeologues that can be helpful for the enhancement of agronomic traits. The CRISPR-Cas9 system provides a promising platform for genome editing in a site-specific manner. In wheat, CRISPR-Cas9 is being used in the improvement of yield, grain quality, biofortification, resistance against diseases, and tolerance against abiotic factors. The promising outcomes of the CRISPR-based multiplexing approach circumvent the constraint of targeting merely one gene at a time. Deployment of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) 9 endonuclease (CRISPR-Cas9) and Cas9 variant systems such as cytidine base editing, adenosine base editing, and prime editing in wheat has been used to induce point mutations more precisely. Scientists have acquired major events such as induction of male sterility, fertility restoration, and alteration of seed dormancy through Cas9 in wheat that can facilitate breeding programs for elite variety development. Furthermore, a recent discovery in tissue culturing enables scientists to significantly enhance regeneration efficiency in wheat by transforming the GRF4-GIF1 cassette. Rapid generation advancement by speed breeding technology provides the opportunity for the generation advancement of the desired plants to segregate out unwanted transgenes and allows rapid integration of gene-edited wheat into the breeding pipeline. The combination of these novel technologies addresses some of the most important limiting factors for sustainable and climate-smart wheat that should lead to the second "Green Revolution" for global food security.

Aegilops tauschii presents a genetic roadmap for hexaploid wheat improvement.

Modern wheat shows phenomenal evolutional success and adaptability to a range of environments owing to polyploidization; however, during its hybridization process a major genetic gain has been overlooked. Recently, Gaurav et al. emphasized harnessing genetic diversity from wheat wild progenitor Aegilops tauschii for the improvement of hexaploid wheat through introgression or transgenesis.

A transgenic approach to control hemipteran insects by expressing insecticidal genes under phloem-specific promoters.

The first generation transgenic crops used strong constitutive promoters for transgene expression. However, tissue-specific expression is desirable for more precise targeting of transgenes. Moreover, piercing/sucking insects, which are generally resistant to insecticidal Bacillus thuringiensis (Bt) proteins, have emerged as a major pests since the introduction of transgenic crops expressing these toxins. Phloem-specific promoters isolated from Banana bunchy top virus (BBTV) were used for the expression of two insecticidal proteins, Hadronyche versuta (Blue Mountains funnel-web spider) neurotoxin (Hvt) and onion leaf lectin, in tobacco (Nicotiana tabacum). Here we demonstrate that transgenic plants expressing Hvt alone or in combination with onion leaf lectin are resistant to Phenacoccus solenopsis (cotton mealybug), Myzus persicae (green peach aphids) and Bemisia tabaci (silver leaf whitefly). The expression of both proteins under different phloem-specific promoters resulted in close to 100% mortality and provided more rapid protection than Hvt alone. Our results suggest the employment of the Hvt and onion leaf lectin transgenic constructs at the commercial level will reduce the use of chemical pesticides for control of hemipteran insect pests.

An unusual abscisic acid and gibberellic acid synergism increases somatic embryogenesis, facilitates its genetic analysis and improves transformation in Medicago truncatula.

Somatic embryogenesis (SE) can be readily induced in leaf explants of the Jemalong 2HA genotype of the model legume Medicago truncatula by auxin and cytokinin, but rarely in wild-type Jemalong. Gibberellic acid (GA), a hormone not included in the medium, appears to act in Arabidopsis as a repressor of the embryonic state such that low ABA (abscisic acid): GA ratios will inhibit SE. It was important to evaluate the GA effect in M. truncatula in order to formulate generic SE mechanisms, given the Arabidopsis information. It was surprising to find that low ABA:GA ratios in M. truncatula acted synergistically to stimulate SE. The unusual synergism between GA and ABA in inducing SE has utility in improving SE for regeneration and transformation in M. truncatula. Expression of genes previously shown to be important in M. truncatula SE was not increased. In investigating genes previously studied in GA investigations of Arabidopsis SE, there was increased expression of GA2ox and decreased expression of PICKLE, a negative regulator of SE in Arabidopsis. We suggest that in M. truncatula there are different ABA:GA ratios required for down-regulating the PICKLE gene, a repressor of the embryonic state. In M. truncatula it is a low ABA:GA ratio while in Arabidopsis it is a high ABA:GA ratio. In different species the expression of key genes is probably related to differences in how the hormone networks optimise their expression.

Cloning and characterization of Na+/H+ antiporter (LfNHX1) gene from a halophyte grass Leptochloa fusca for drought and salt tolerance.

Abiotic stresses such as salinity and drought have adverse effects on plants. In the present study, a Na(+)/H(+) antiporter gene homologue (LfNHX1) has been cloned from a local halophyte grass (Leptochloa fusca). The LfNHX1 cDNA contains an open reading frame of 1,623 bp that encodes a polypeptide chain of 540 amino acid residues. LfNHX1 protein sequence showed high similarity with NHX1 homologs reported from other halophyte plants. Amino acid and nucleotide sequence similarity, protein topology modeling and the presence of conserved functional domains in the LfNHX1 protein sequence classified it as a vacuolar NHX1 homolog. The overexpression of LfNHX1 gene under CaMV35S promoter conferred salt and drought tolerance in tobacco plants. Under drought stress, transgenic plants showed higher relative water contents, photosynthetic rate, stomatal conductance and membrane stability index as compared to wild type plants. More negative value of leaf osmotic potential was also observed in transgenic plants when compared with wild type control plants. Transgenic plants showed better germination and root growth at 2 mg L(-1) Basta herbicide and three levels (100, 200 and 250 mM) of sodium chloride. These results showed that LfNHX1 is a potential candidate gene for enhancing drought and salt tolerance in crops.

The transcription factor MtSERF1 of the ERF subfamily identified by transcriptional profiling is required for somatic embryogenesis induced by auxin plus cytokinin in Medicago truncatula.

Transcriptional profiling of embryogenic callus produced from Medicago truncatula mesophyll protoplasts indicated up-regulation of ethylene biosynthesis and ethylene response genes. Using inhibitors of ethylene biosynthesis and perception, it was shown that ethylene was necessary for somatic embryogenesis (SE) in this model legume. We chose several genes involved in ethylene biosynthesis and response for subsequent molecular analyses. One of these genes is a gene encoding a transcription factor that belongs to the AP2/ERF superfamily and ERF subfamily of transcription factors. We demonstrate that this gene, designated M. truncatula SOMATIC EMBRYO RELATED FACTOR1 (MtSERF1), is induced by ethylene and is expressed in embryogenic calli. MtSERF1 is strongly expressed in the globular somatic embryo and there is high expression in a small group of cells in the developing shoot meristem of the heart-stage embryo. RNA interference knockdown of this gene causes strong inhibition of SE. We also provide evidence that MtSERF1 is expressed in zygotic embryos. MtSERF1 appears to be essential for SE and may enable a connection between stress and development.

The stress kinase gene MtSK1 in Medicago truncatula with particular reference to somatic embryogenesis.

Medicago truncatula, a model for legume genomics, can be regenerated by somatic embryogensis by the use of a suitable genotype and an auxin plus cytokinin. The stress response induced by explant wounding and culture is increasingly recognized as an important component of somatic embryo induction. We have cloned and investigated the stress kinase gene MtSK1 in relation to somatic embryogenesis in M. truncatula, using the highly embryogenic mutant Jemalong 2HA (2HA) and its progenitor Jemalong. The main features of the MtSK1 protein of 351 amino acids are an N-terminal kinase domain and a C-terminal glutamic acid-rich region, which is predicted to be a coiled-coil. MtSK1 is a member of the SnRK2 subgroup of the SnRK group of plant kinases. Members of the SnRK2 kinases play a role in stress responses of plants. MtSKI expression is induced by wounding in the cultured tissue independent of auxin or cytokinin. However, in both 2HA and Jemalong, as the callus develops in response to auxin plus cytokinin, MtSK1 expression continues to increase. MtSK1 responds to salt stress in vivo, consistent with its role as a stress kinase. The likely role of MtSK1 in stress-induced signaling will facilitate the relating of stress-response pathways to auxin and cytokinin-induced signaling in the understanding of the molecular mechanisms involved in the induction of somatic embryogenesis in M. truncatula.

Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts.

Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.