Dissecting the genomic landscape of ERD genes in alfalfa (Medicago sativa): the effect of melatonin and low selenium under salt stress.

Light-harvesting chlorophyll a/b-binding proteins (LHC) of photosystem II perform key functions in various processes, e.g., photosynthesis, development, and abiotic stress responses. Nonetheless, comprehensive genome-wide investigation of LHC family genes (CrLHCs) has not been well-reported in single-cell alga (Chlamydomonas reinhardtii). Here, we discovered 61 putative CrLHC genes in the C. reinhardtii genome and observed that most genes demonstrate stable exon-intron and motif configurations. We predicted five phytohormones- and six abiotic stress-interrelated cis-regulatory elements in promoter regions of CrLHC. Likewise, 19 miRNAs targeting 42 CrLHC genes from 16 unique families were discovered. Besides, we identified 400 transcription factors from 13 families, including ERF, GATA, CPP, bZIP, C3H, MYB, SBP, Dof, bHLH, C2H2, G2-like, etc. Protein-protein interactions and 3D structures provided insight into CrLHC proteins. Gene ontology and KEGG-based enrichment advocated their role in light responses, photosynthesis, and energy metabolisms. Expression analysis highlighted the shared and unique roles of many CrLHC genes against different abiotic stresses (UV-C, green light, heat, nitric oxide, cadmium, nitrogen starvation, and salinity). Under salinity stress, antioxidant enzyme activity, reactive oxygen species markers, photosynthesis-related traits and pigments were significantly affected. Briefly, this comprehensive genomic and physiological study shed light on the impact of CrLHC genes in abiotic stress tolerance and set the path for future genetic engineering experiments.

SQUAMOSA Promoter-binding protein-Like (SPL) genes affect a broad range of plant biological processes and show potential application in crop improvement by genetic modification. As the most widely planted forage crop in the world, biomass and abiotic stresses tolerance are important breeding targets for alfalfa (Medicago sativa L.). Nevertheless, the systematic analysis of SPL genes in alfalfa genome remains lacking. In the present study, we characterized 22 putative non-redundant SPL genes in alfalfa genome and uncovered the abundant structural variation among MsSPL genes. The phylogenetic analysis of plant SPL proteins separated them into 10 clades and clade J was an alfalfa-specific clade, suggesting SPL genes in alfalfa might have experienced gene duplication and functional differentiation within the genome. Meanwhile, 11 MsSPL genes with perfect matches to miRNA response elements (MREs) could be degraded by miR156, and the cleavage sites were gene specific. In addition, we investigated the temporal and spatial expression patterns of MsSPL genes and their expression patterns in response to multiple treatments, characterizing candidate SPL genes in alfalfa development and abiotic stress tolerant regulation. More importantly, overexpression of the alfalfa-specific SPL gene (MsSPL20) showed stable delayed flowering time, as well as increased biomass. Further studies indicated that MsSPL20 delayed flowering time by regulating the expression of genes involved in floret development, including HD3A, FTIP1, TEM1, and HST1. Together, our findings provide valuable information for future research and utilization of SPL genes in alfalfa and elucidate a possibly alfalfa-specific flowering time regulation, thereby supplying candidate genes for alfalfa molecular-assisted breeding.

Identification of candidate gene(s) and its validation in a breeder’s germplasm is a prerequisite for any successful marker-assisted selection (MAS) programme for improving abiotic stress tolerance. Once a candidate gene(s) is identified and its effects validated under a stress environment, it becomes a powerful marker resource for developing ‘functional markers’ to assist genomics-assisted breeding in crops. There are several ways to identify a candidate gene(s) underpinning a specific abiotic stress tolerance mechanism. The most common methods used are various ‘omics’ approaches targeting transcriptome (transcriptomics), metabolome (metabolomics) or proteome (proteomics), co-location of genes with quantitative trait loci (QTLs) for abiotic stress tolerance traits (called positional candidates), fine mapping of QTLs/QTL cloning, transgenics, RNA interference, mutant screenings and genome wide/candidate gene-based association mapping among others. The advent of next generation sequencing (NGS) technologies has completely revolutionized the identification and characterization of candidate genes underlying various abiotic stress tolerance traits. This review focuses on the approaches taken to identify and validate candidate genes for various abiotic stress tolerances in wheat and the progress made so far in their validation and implementation in wheat breeding programs globally.

Progress in genetic analysis and breeding of tepary bean (Phaseolus acutifolius A. Gray): A review

The S1fa transcription factor is part of a small family involved in plant growth and development and abiotic stress tolerance. However, the roles of the S1fa genes in abiotic stress tolerance in Chinese cabbage are still unclear. In this study, four S1fa genes in the Chinese cabbage genome were identified and characterized for abiotic stress tolerance. Tissue-specific expression analysis suggested that three of these four S1fa genes were expressed in all tissues of Chinese cabbage, while Bra006994 was only expressed in the silique. Under Hg and Cd stresses, the S1fa genes were significantly expressed but were downregulated under NaCl stresses. The Bra034084 and Bra029784 overexpressing yeast cells exhibited high sensitivity to NaCl stresses, which led to slower growth compared with the wild type yeast cells (EV) under 1 M NaCl stress. In addition, the growth curve of the Bra034084 and Bra029784 overexpressing cells shows that the optical density was reduced significantly under salt stresses. The activities of the antioxidant enzymes, SOD, POD and CAT, were decreased, and the MDA, H2O2 and O2− contents were increased under salt stresses. The expression levels of cell wall biosynthesis genes Ccw14p, Cha1p, Cwp2p, Sed1p, Rlm1p, Rom2p, Mkk1p, Hsp12p, Mkk2p, Sdp1p and YLR194c were significantly enhanced, while Bck1p, and Ptc1p were downregulated under salt stresses. These results suggest that the Bra034084 and Bra029784 genes regulate cell wall biosynthesis and the defense regulatory system under salt stresses. These findings provide a fundamental basis for the further investigation of crop genetic modification to improve crop production and abiotic stress tolerance in Chinese cabbage.

Rice (Oryza sativa L) feeds more than half of the world’s population. One of the main elements that harm yield globally is abiotic stress. Therefore, it is important to develop abiotic stress tolerant rice varieties in order to increase rice productivity and to extend the cultivation. The lack of knowledge of the genetic mechanisms underlying abiotic stress tolerance is the primary issue with the traditional breeding technique. Hence, studying genes responsible for abiotic stress mechanisms is important to accelerate breeding by molecular marker - based detection techniques. Aiming at finding the candidate genes for abiotic stress tolerance ,two rice genome sequences of At 354 and Bg 352 varieties given by National Research Council, Sri Lanka -16 -16 project were analyzed. At 354 has some abiotic stress tolerance (salt) traits and Bg 352 has some susceptible traits. Next-generation sequencing-derived genome sequences were used to identify SNPs and Indels in the At 354 and Bg 352 varieties with reference to Oryza sativa japonica group cultivar Nipponbare. The STRING Database was used to extract the most correlated genes with abiotic stress. The allelic differences among Nipponbare, At 354 and Bg 352 sequences were detected from Multiple Sequence Alignment by using the Rice Annotation Project database, UGENE software and MEGA 11 software. The mutations of the genes were validated if they were present in another germplasm in the NCBI database. Altogether 100 genes were used to examine, and 166 mutations were observed including 163 SNPs and 3 Indels while 09 genes were validated due to their presence in other rice accessions. The amino acid sequences of the validated sequences were determined by Expasy Translate tool. The Swiss model database and ProtParam tool were used to predict the protein structures and their parameters, which showed some structural differences among tested alleles. These mutant alleles further need to be assessed against abiotic stress and varietal turnover in order to use them in rice improving breeding programs to be used in abiotic stress-prone ecosystems.

Small millets, a member of the Poaceae family grows well in Asia, Africa, and some regions of Europe. These millets are a great source of protein, therefore, it is highly used as a source of food for humans, animals, and birds. Despite the presence of higher content of protein, it is also rich in other essential nutrients such as vitamins, minerals, and, dietary fibers. These millets exhibit biological properties such as being anti-inflammatory, anti-cancerous, and lowering cholesterol and glucose levels in the body. Even though millets exhibit significant biological functions, their consumption and production are declining globally. This may be due to easy availability and easier cooking methods of other prominent cereals such as rice, wheat, and maize. Small millets are also widely used as a potent source for the production of starch and alcohol which is escalating the demand for these millets. The production of small millets is mainly affected due to biotic and abiotic factors. Biotic factors include fungal, bacterial, and viral infections whereas drought, salinity, waterlogging, and lodging are the abiotic factors that greatly affect their production and yield. Reliable and robust methods of plant regeneration, identification of novel functional genes responsible for abiotic stress tolerance, and introduction of new traits to small millets by establishing Agrobacterium-mediated transformation have paved the way for the development of abiotic stress-tolerant millets. This chapter highlights different biotic and abiotic factors that inhibit the growth of small millets, various plant regeneration methods, transformation studies, potential genes for abiotic stress tolerance, and transgenic approaches for the production of improved abiotic stress-tolerant millets.KeywordsSmall milletsBiotic stressAbiotic stressPlant regenerationTransformationStress tolerance

Salinity stress is one of the major factors which reduce crop plants growth and productivity resulting in significant economic losses worldwide. Therefore, it would be fruitful to isolate and functionally identify new salinity stress-induced genes for understanding the mechanism and developing salinity stress tolerant plants. Based on functional gene screening assay, we have isolated three salinity tolerant genes out of one million Escherichia coli (SOLR) transformants containing pea cDNAs. Sequence analysis of three of these genes revealed homology to Ribosomal-L30E (RPL30E), Chlorophyll-a/b-binding protein (Chla/bBP) and FIDDLEHEAD (FDH). The salinity tolerance of these genes in bacteria was further confirmed by using another strain of E. coli (DH5α) transformants. The homology based computational modeling of these proteins suggested the high degree of conservation with the conserved domains of their homologous partners. The reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression of these cDNAs (except the FDH) was upregulated in pea plants in response to NaCl stress. We observed that there was no significant effect of Li+ ion on the expression level of these genes, while an increase in response to K+ ion was observed. Overall, this study provides an evidence for a novel function of these genes in high salinity stress tolerance. The PsFDH showed constitutive expression in planta suggesting that it can be used as constitutively expressed marker gene for salinity stress tolerance in plants. This study brings new direction in identifying novel function of unidentified genes in abiotic stress tolerance without previous knowledge of the genome sequence.

Abiotic stresses including drought, salinity and cold are a major challenge for sustainable food production as they may decrease the potential yields in crop plants by 70 %. Success in breeding for better adapted varieties to abiotic stresses depends upon intensive efforts using novel biotechnological approaches, including molecular biology, genetics, plant and cell physiology and breeding. Many abiotic stress-induced genes have been identified and some have been cloned. The use of current molecular biology tools to reveal the control mechanisms of abiotic-stress tolerance, and for engineering stress-tolerant crops is based on the expression of specific stress-related genes. Hence, plant genetic engineering and molecular-marker approaches allow development of abiotic stress-tolerant germplasm. Transgenic plants carrying genes for abiotic stress tolerance are being developed, mainly by using Agrobacterium and biolistic methods; transgenics carrying different genes relating to abiotic stress tolerance have been developed in crop plants like rice, wheat, maize, sugarcane, tobacco, Arabidopsis, groundnut, tomato and potato. This chapter focuses on recent progress in using transgenic technology for the improvement of abiotic-stress tolerance in plants. It includes discussion of metabolic engineering for biosynthesis and accumulation of compatible osmolytes (i.e. proline, glycine betaine, ectoine and polyols), reactive oxygen species formation under abiotic stress, ROS scavenging and detoxification in plant cells, single gene transgenic versus multiple genes and transcription factors and their roles in management of abiotic stresses.

Crop yield, survival, and biomass production are negatively influenced by abiotic stresses. Due to multigenic nature, the molecular basis of abiotic stress tolerance is difficult to understand. Modern agriculture faces various challenges which include global climate change, complex field environment, and the combination of abiotic stress. To improve abiotic tolerance in crop plants, a combined effect of various molecular and biochemical approaches will be needed. Advanced molecular biology tools are used to enlighten the regulation mechanism of abiotic stress tolerance based on expression analysis of various stress-linked genes. The data collected from high-throughput transcription profiling, identification of specific protein network on large scale, molecular modeling and their association with environmental changes in plants all reveal information about plant system which is used for engineering plants against various environmental changes. Various genes for abiotic stress tolerance in crop plants have been identified and cloned to develop stress tolerant plants. In spite of various advancements in the technology, the success in developing stress tolerant plants is limited. This review enlightens the recent advancement in transgenic technology for the betterment of crop plants against abiotic stress.

Agricultural productivity is majorly impacted due to various abiotic stresses, particularly salinity and drought. Halophytes serve as an excellent resource for identifying and developing new crop systems, as these grow very luxuriously in very high saline soils. Understanding salinity stress tolerance mechanisms in such plants is an important step towards generating crop varieties that can cope with environmental stresses. Use of modern tools of ‘omics’ analyses and small RNA sequencing has helped to gain insights into the complex plant stress responses. Salinity tolerance being a multigenic trait requires a combination of strategies and techniques to successfully develop improved crops varieties. Many transgenic crops are being developed through genetic transformation. Besides marker-assisted breeding/QTL approaches are also being used to improve abiotic stress tolerance. In this review, we focus on the recent developments in the utilization of halophytes as a source of genes for genetic improvement in abiotic stress tolerance of crops.

Sorghum bicolor (L.) Moench is a multipurpose food crop which is ranked among the top five cereal crops in the world, and is used as a source of food, fodder, feed, and fuel. The genus Sorghum consists of 24 diverse species. Cultivated sorghum was derived from the wild progenitor S. bicolor subsp. verticilliflorum, which is commonly distributed in Africa. Archeological evidence has identified regions in Sudan, Ethiopia, and West Africa as centers of origin of sorghum, with evidence for more than one domestication event. The taxonomy of the genus is not fully resolved, with alternative classifications that should be resolved by further molecular analysis. Sorghum can withstand severe droughts which makes it suitable to grow in regions where other major crops cannot be grown. Wild relatives of many crops have played significant roles as genetic resources for crop improvement. Although there have been many studies of domesticated sorghum, few studies have reported on its wild relatives. In Sorghum, some species are widely distributed while others are very restricted. Of the 17 native sorghum species found in Australia, none have been cultivated. Isolation of these wild species from domesticated crops makes them a highly valuable system for studying the evolution of adaptive traits such as biotic and abiotic stress tolerance. The diversity of the genus Sorghum has probably arisen as a result of the extensive variability of the habitats over which they are distributed. The wild gene pool of sorghum may, therefore, harbor many useful genes for abiotic and biotic stress tolerance. While there are many examples of successful examples of introgression of novel alleles from the wild relatives of other species from Poaceae, such as rice, wheat, maize, and sugarcane, studies of introgression from wild sorghum are limited. An improved understanding of wild sorghums will better allow us to exploit this previously underutilized gene pool for the production of more resilient crops.

Transgenic plants with improved salt and drought stress tolerance have been developed with a large number of abiotic stress-related genes. Among these, the most extensively used genes are the glycine betaine biosynthetic codA, the DREB transcription factors, and vacuolar membrane Na+/H+ antiporters. The use of codA, DREBs, and Na+/H+ antiporters in transgenic plants has conferred stress tolerance and improved plant phenotype. However, the future deployment and commercialization of these plants depend on their safety to the environment. Addressing environmental risk assessment is challenging since mechanisms governing abiotic stress tolerance are much more complex than that of insect resistance and herbicide tolerance traits, which have been considered to date. Therefore, questions arise, whether abiotic stress tolerance genes need additional considerations and new measurements in risk assessment and, whether these genes would have effects on weediness and invasiveness potential of transgenic plants? While considering these concerns, the environmental risk assessment of abiotic stress tolerance genes would need to focus on the magnitude of stress tolerance, plant phenotype and characteristics of the potential receiving environment. In the present review, we discuss environmental concerns and likelihood of concerns associated with the use of abiotic stress tolerance genes. Based on our analysis, we conclude that the uses of these genes in domesticated crop plants are safe for the environment. Risk assessment, however, should be carefully conducted on biofeedstocks and perennial plants taking into account plant phenotype and the potential receiving environment.

BackgroundDrought stress is a predominant abiotic factor contributing to reduced crop yields globally. Therefore, exploring the molecular mechanism of drought control is of great significance to improve drought resistance and ultimately achieve crop yield increase. As a plant endogenous hormone, melatonin plays a key role in the regulation of abiotic stress, but the key genes and metabolic pathways of melatonin mediated drought resistance regulation in alfalfa have not been fully revealed. Based on transcriptomics and physiological index detection, this study aimed to explore the regulatory mechanism of melatonin in alleviating drought stress during alfalfa germination.ResultsThe findings revealed that alfalfa seedlings treated with melatonin exhibited higher germination rates, increased shoot length, and greater fresh weight compared to those exposed solely to drought stress. Additionally, there was a reduction in the levels of malondialdehyde (MDA) and superoxide anion (O2−), while the activity and concentration of superoxide dismutase (SOD), peroxidase (POD), and glutathione (GSH) were enhanced to varying extents. To investigate the molecular mechanism underlying melatonin-mediated drought resistance in alfalfa, we performed a transcriptomic analysis on the seedlings. In the drought treatment group, we identified a total of 1,991 differentially expressed genes (DEGs), comprising 778 up-regulated and 1,213 down-regulated genes. Conversely, in the melatonin-treated group, we discovered 2,336 DEGs, including 882 up-regulated and 1,454 down-regulated genes.ConclusionsThrough the application of GO functional annotation and KEGG pathway enrichment analysis, we discovered that DEGs were predominantly enriched in pathways related to flavonoid and isoflavone biosynthesis, plant hormone biosynthesis and signal transduction, glutathione metabolism, and MAPK signaling, and the ABC transporter signaling. Notably, the DEGs added to the MT group showed greater enrichment in these pathways. This suggests that MT mitigates drought stress by modulating the expression of genes associated with energy supply and antioxidant capacity. These findings hold significant reference value for breeding drought-tolerant alfalfa and other crops.

Drought is the most crucial environmental factor that limits productivity of many crop plants. Exploring novel genes and gene combinations is of primary importance in plant drought tolerance research. Stress tolerant genotypes/species are known to express novel stress responsive genes with unique functional significance. Hence, identification and characterization of stress responsive genes from these tolerant species might be a reliable option to engineer the drought tolerance. Safflower has been found to be a relatively drought tolerant crop and thus, it has been the choice of study to characterize the genes expressed under drought stress. In the present study, we have evaluated differential drought tolerance of two cultivars of safflower namely, A1 and Nira using selective physiological marker traits and we have identified cultivar A1 as relatively drought tolerant. To identify the drought responsive genes, we have constructed a stress subtracted cDNA library from cultivar A1 following subtractive hybridization. Analysis of ~1,300 cDNA clones resulted in the identification of 667 unique drought responsive ESTs. Protein homology search revealed that 521 (78 %) out of 667 ESTs showed significant similarity to known sequences in the database and majority of them previously identified as drought stress-related genes and were found to be involved in a variety of cellular functions ranging from stress perception to cellular protection. Remaining 146 (22 %) ESTs were not homologous to known sequences in the database and therefore, they were considered to be unique and novel drought responsive genes of safflower. Since safflower is a stress-adapted oil-seed crop this observation has great relevance. In addition, to validate the differential expression of the identified genes, expression profiles of selected clones were analyzed using dot blot (reverse northern), and northern blot analysis. We showed that these clones were differentially expressed under different abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance are discussed in our study.