-
Plant Molecular Biology Apr 2024The GATA transcription factors (TFs) have been extensively studied for its regulatory role in various biological processes in many plant species. The functional and...
The GATA transcription factors (TFs) have been extensively studied for its regulatory role in various biological processes in many plant species. The functional and molecular mechanism of GATA TFs in regulating tolerance to abiotic stress has not yet been studied in the common bean. This study analyzed the functional identity of the GATA gene family in the P. vulgaris genome under different abiotic and phytohormonal stress. The GATA gene family was systematically investigated in the P. vulgaris genome, and 31 PvGATA TFs were identified. The study found that 18 out of 31 PvGATA genes had undergone duplication events, emphasizing the role of gene duplication in GATA gene expansion. All the PvGATA genes were classified into four significant subfamilies, with 8, 3, 6, and 13 members in each subfamily (subfamilies I, II, III, and IV), respectively. All PvGATA protein sequences contained a single GATA domain, but subfamily II members had additional domains such as CCT and tify. A total of 799 promoter cis-regulatory elements (CREs) were predicted in the PvGATAs. Additionally, we used qRT-PCR to investigate the expression profiles of five PvGATA genes in the common bean roots under abiotic conditions. The results suggest that PvGATA01/10/25/28 may play crucial roles in regulating plant resistance against salt and drought stress and may be involved in phytohormone-mediated stress signaling pathways. PvGATA28 was selected for overexpression and cloned into N. benthamiana using Agrobacterium-mediated transformation. Transgenic lines were subjected to abiotic stress, and results showed a significant tolerance of transgenic lines to stress conditions compared to wild-type counterparts. The seed germination assay suggested an extended dormancy of transgenic lines compared to wild-type lines. This study provides a comprehensive analysis of the PvGATA gene family, which can serve as a foundation for future research on the function of GATA TFs in abiotic stress tolerance in common bean plants.
Topics: Phaseolus; GATA Transcription Factors; Agrobacterium; Amino Acid Sequence; Droughts; Plant Growth Regulators
PubMed: 38630371
DOI: 10.1007/s11103-024-01443-y -
PloS One 2024Most legumes are able to develop a root nodule symbiosis in association with proteobacteria collectively called rhizobia. Among them, the tropical species Aeschynomene...
Setting up Agrobacterium tumefaciens-mediated transformation of the tropical legume Aeschynomene evenia, a powerful tool for studying gene function in Nod Factor-independent symbiosis.
Most legumes are able to develop a root nodule symbiosis in association with proteobacteria collectively called rhizobia. Among them, the tropical species Aeschynomene evenia has the remarkable property of being nodulated by photosynthetic Rhizobia without the intervention of Nod Factors (NodF). Thereby, A. evenia has emerged as a working model for investigating the NodF-independent symbiosis. Despite the availability of numerous resources and tools to study the molecular basis of this atypical symbiosis, the lack of a transformation system based on Agrobacterium tumefaciens significantly limits the range of functional approaches. In this report, we present the development of a stable genetic transformation procedure for A. evenia. We first assessed its regeneration capability and found that a combination of two growth regulators, NAA (= Naphthalene Acetic Acid) and BAP (= 6-BenzylAminoPurine) allows the induction of budding calli from epicotyls, hypocotyls and cotyledons with a high efficiency in media containing 0,5 μM NAA (up to 100% of calli with continuous stem proliferation). To optimize the generation of transgenic lines, we employed A. tumefaciens strain EHA105 harboring a binary vector carrying the hygromycin resistance gene and the mCherry fluorescent marker. Epicotyls and hypocotyls were used as the starting material for this process. We have found that one growth medium containing a combination of NAA (0,5 μM) and BAP (2,2 μM) was sufficient to induce callogenesis and A. tumefaciens strain EHA105 was sufficiently virulent to yield a high number of transformed calli. This simple and efficient method constitutes a valuable tool that will greatly facilitate the functional studies in NodF-independent symbiosis.
Topics: Fabaceae; Agrobacterium tumefaciens; Symbiosis; Phenotype; Vegetables; Transformation, Genetic; Plants, Genetically Modified
PubMed: 38625963
DOI: 10.1371/journal.pone.0297547 -
Microbiology Spectrum Jun 2024We conducted a comprehensive analysis of the total microbiome and transcriptionally active microbiome communities in the roots and root nodules of , an important...
We conducted a comprehensive analysis of the total microbiome and transcriptionally active microbiome communities in the roots and root nodules of , an important leguminous tree in arid regions of many Asian countries. Mature trees growing in the desert did not exhibit any detected root nodules. However, we observed root nodules on the roots of growing on a desert farm and on young plants growing in a growth chamber, when inoculated with rhizosphere soil, including with rhizosphere soil from near desert tree roots that had no nodules. Compared to nearby soil, non-nodulated roots were enriched with Actinobacteria (e.g., sp.), whereas root nodules sampled from the desert farm and growth chamber had abundant Alphaproteobacteria (e.g., sp.). These nodules yielded many microbes in addition to such nitrogen-fixing bacteria as and species. Significant differences exist in the composition and abundance of microbial isolates between the nodule surface and the nodule endosphere. Shotgun metagenome analysis of nodule endospheres revealed that the root nodules comprised over 90% bacterial DNA, whereas metatranscriptome analysis showed that the plant produces vastly more transcripts than the microbes in these nodules. Control inoculations demonstrated that four out of six or isolates purified from nodules produced nodules in the roots of seedlings under greenhouse conditions. The best nodulation was achieved when seedlings were inoculated with a mixture of those bacterial strains. Though root nodulation could be achieved under water stress conditions, nodule number and nodule biomass increased with copious water availability. .IMPORTANCEMicrobial communities were investigated in roots and root nodules of , a leguminous tree species in arid Asian regions that is responsible for exceptionally important contributions to soil fertility in these dramatically dry locations. Soil removed from regions near nodule-free roots on these mature plants contained an abundance of bacteria with the genetic ability to generate nodules and fix nitrogen but did not normally nodulate in their native rhizosphere environment, suggesting a very different co-evolved relationship than that observed for herbaceous legumes. The relative over-expression of the low-gene-density plant DNA compared to the bacterial DNA in the nodules was also unexpected, indicating a very powerful induction of host genetic contributions within the nodule. Finally, the water dependence of nodulation in inoculated seedlings suggested a possible link between early seedling growth (before a deep root system can be developed) and the early development of nitrogen-fixing capability.
Topics: Microbiota; Root Nodules, Plant; Soil Microbiology; Prosopis; Desert Climate; Rhizosphere; Plant Roots; Bacteria; Symbiosis; Trees; Nitrogen Fixation; Phylogeny
PubMed: 38624222
DOI: 10.1128/spectrum.03617-23 -
Bio-protocol Apr 2024Citrus fruits encompass a diverse family, including oranges, mandarins, grapefruits, limes, kumquats, lemons, and others. In citrus, -mediated genetic transformation of...
Citrus fruits encompass a diverse family, including oranges, mandarins, grapefruits, limes, kumquats, lemons, and others. In citrus, -mediated genetic transformation of Hongkong kumquat ( Swingle) has been widely employed for gene function analysis. However, the perennial nature of woody plants results in the generation of transgenic fruits taking several years. Here, we show the procedures of -mediated transient transformation and live-cell imaging in kumquat ( Swingle) fruit, using the actin filament marker GFP-Lifeact as an example. Fluorescence detection, western blot analysis, and live-cell imaging with confocal microscopy demonstrate the high transformation efficiency and an extended expression window of this system. Overall, -mediated transient transformation of kumquat fruits provides a rapid and effective method for studying gene function in fruit, enabling the effective observation of diverse cellular processes in fruit biology.
PubMed: 38618180
DOI: 10.21769/BioProtoc.4968 -
Bio-protocol Apr 2024, a Brassicaceae family crop, is used for fodder, human food, and biofuels. Its relatively high resistance to abiotic and biotic stresses, as well as being a...
, a Brassicaceae family crop, is used for fodder, human food, and biofuels. Its relatively high resistance to abiotic and biotic stresses, as well as being a climate-resilient oilseed crop, has contributed to its popularity. Camelina's seed yield and oil contents have been improved using various technologies like RNAi and CRISPR/Cas9 genome editing. A stable transformation system for protein localization and other cell autonomous investigations, on the other hand, is tedious and time consuming. This study describes a transient gene expression protocol for Camelina sativa cultivar DH55 leaves using Agrobacterium strain C58C1. The method is suitable for subcellular protein localization and colocalization studies and can be used with both constitutive and chemically induced genes. We report the subcellular localization of the N-terminal ER membrane signal anchor region (1-32 aa) of the At3G28580 gene-encoded protein from Arabidopsis in intact leaves and the expression and localization of other known organelle markers. This method offers a fast and convenient way to study proteins in the commercially important Camelina crop system. Key features • This method is based on the approach of Zhang et al. [1] and has been optimized for bioenergy crop species. • A constitutive and inducible transient gene expression in the hexaploid species cultivar DH55. • Requires only 16-18 days to complete with high efficacy. Graphical overview.
PubMed: 38618179
DOI: 10.21769/BioProtoc.4964 -
Revista Argentina de Microbiologia Apr 2024Phytopathogenic fungi Alternaria alternata and Colletotrichum gloeosporioides cause diseases in plant tissues as well as significant postharvest losses. The use of...
Phytopathogenic fungi Alternaria alternata and Colletotrichum gloeosporioides cause diseases in plant tissues as well as significant postharvest losses. The use of chemical fungicides for their control has negative effects on health and the environment. Secondary metabolites from halophilic bacteria are a promising alternative for new antifungal compounds. In the present study, halophilic bacteria were isolated and characterized from two sites with saline soils called branquizales in Campeche, Mexico. A total of 64 bacteria were isolated. Agrobacterium, Bacillus, Inquilinus, Gracilibacillus, Metabacillus, Neobacillus, Paenibacillus, Priestia, Staphylococcus, Streptomyces and Virgibacillus were among the identified genera. The antifungal potential of the culture supernatant (CS) of 39 halophilic bacteria was investigated against C. gloeosporioides and A. alternata. The bacteria showing the greatest inhibition of mycelial growth corresponded to Bacillus subtilis CPO 4292, Metabacillus sp. CPO 4266, Bacillus sp. CPO 4295 and Bacillus sp. CPO 4279. The CS of Bacillus sp. CPO 4279 exhibited the highest activity and its ethyl acetate extract (AcOEt) inhibited the germination of C. gloeosporioides, with IC values of 8,630μg/ml and IC of 10,720μg/ml. The organic partition of the AcOEt extract led to three fractions, with acetonitrile (FAcB9) showing the highest antifungal activity, with values exceeding 66%. Halophilic bacteria from 'blanquizales' soils of the genus Bacillus sp. produce metabolites with antifungal properties that inhibit the phytopathogenic fungus C. gloeosporioides.
PubMed: 38614909
DOI: 10.1016/j.ram.2024.02.004 -
Plants (Basel, Switzerland) Apr 2024This study aims to establish an -mediated transformation system for use with the 'MiniMax'soybean cultivar. MiniMax is a mutant soybean whose growth cycle is around 90...
This study aims to establish an -mediated transformation system for use with the 'MiniMax'soybean cultivar. MiniMax is a mutant soybean whose growth cycle is around 90 days, half that of most other soybean varieties, making it an optimal model cultivar to test genes of interest before investing in modification of elite lines. We describe an efficient protocol for -mediated transformation using MiniMax seeds. It uses a modified 'half seed' regeneration protocol for transgenic soybean production, utilizing the rapid generation MiniMax variety to obtain T1 seeds in approximately 145 days. Addition of phloroglucinol (PG) to the regeneration protocol was key to obtaining high-efficiency rooting of the regenerated shoots. Transfer to soil was accomplished using an organic soil amendment containing nutrients and mycorrhiza for plants to thrive in the greenhouse. This combination of genotype and stimulants provides a transformation protocol to genetically engineer MiniMax seeds with a transgenic lab-to-greenhouse production efficiency of 4.0%. This is the first report of MiniMax soybean whole plant transformation and heritable T1 transmission. This protocol provides an ideal resource for enhancing the genetic transformation of any soybean cultivar.
PubMed: 38611542
DOI: 10.3390/plants13071013 -
Frontiers in Plant Science 2024Under changing climatic scenarios, grassland conservation and development have become imperative to impart functional sustainability to their ecosystem services. These... (Review)
Review
Under changing climatic scenarios, grassland conservation and development have become imperative to impart functional sustainability to their ecosystem services. These goals could be effectively and efficiently achieved with targeted genetic improvement of native grass species. To the best of our literature search, very scant research findings are available pertaining to gene editing of non-cultivated grass species (switch grass, wild sugarcane, Prairie cordgrass, Bermuda grass, Chinese silver grass, etc.) prevalent in natural and semi-natural grasslands. Thus, to explore this novel research aspect, this study purposes that gene editing techniques employed for improvement of cultivated grasses especially sugarcane might be used for non-cultivated grasses as well. Our hypothesis behind suggesting sugarcane as a model crop for genetic improvement of non-cultivated grasses is the intricacy of gene editing owing to polyploidy and aneuploidy compared to other cultivated grasses (rice, wheat, barley, maize, etc.). Another reason is that genome editing protocols in sugarcane ( = 10-13) have been developed and optimized, taking into consideration the high level of genetic redundancy. Thus, as per our knowledge, this review is the first study that objectively evaluates the concept and functioning of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technique in sugarcane regarding high versatility, target specificity, efficiency, design simplicity, and multiplexing capacity in order to explore novel research perspectives for gene editing of non-cultivated grasses against biotic and abiotic stresses. Additionally, pronounced challenges confronting sugarcane gene editing have resulted in the development of different variants (Cas9, Cas12a, Cas12b, and SpRY) of the CRISPR tool, whose technicalities have also been critically assessed. Moreover, different limitations of this technique that could emerge during gene editing of non-cultivated grass species have also been highlighted.
PubMed: 38601306
DOI: 10.3389/fpls.2024.1369416 -
Plants (Basel, Switzerland) Mar 2024(), belonging to the / gene family, is an important gene regulating the flowering transition and inflorescence architecture during plant development. Given its...
(), belonging to the / gene family, is an important gene regulating the flowering transition and inflorescence architecture during plant development. Given its importance to plant adaptation and crop improvement, has been extensively studied in related plant research; however, the specific role and underlying molecular mechanisms of in the continuous flowering of perennial plants remains elusive. Here, we isolated and characterized homologous genes from two species with different flowering-period phenotypes: was isolated from , a precious species blooming in summer and flowering throughout the year, and was isolated from , which blooms in winter and spring. The major difference in the genes between the two species was an additional five-amino acid repeat sequence in . showed high expression levels in the leaves in both species from January to August, especially in April for and in May for . was expressed throughout the year in , whereas was not expressed from September to December in . The expression levels of in the floral buds were generally higher than those in the leaves. Overexpression of and in indicated that both genes can activate downstream genes to promote flowering. Transgenic callus tissue was obtained by introducing the two genes into through -mediated transformation. Transcriptome and quantitative real-time polymerase chain reaction analyses indicated that both florigen genes promoted the expression of downstream genes such as , , and , and slightly up-regulated the expression of upstream genes such as and . The above results indicated that and played a role in promoting flowering in both camellia species. The expression pattern of in leaves suggested that, compared to may be related to the annual flowering of .
PubMed: 38592966
DOI: 10.3390/plants13060784 -
Plants (Basel, Switzerland) Mar 2024Sugarcane ( spp.), a major cash crop that is an important source of sugar and bioethanol, is strongly influenced by the impacts of biotic and abiotic stresses. The...
Sugarcane ( spp.), a major cash crop that is an important source of sugar and bioethanol, is strongly influenced by the impacts of biotic and abiotic stresses. The intricate polyploid and aneuploid genome of sugarcane has shown various limits for conventional breeding strategies. Nonetheless, biotechnological engineering currently offers the best chance of introducing commercially significant agronomic features. In this study, an efficient -mediated transformation system that uses the herbicide-resistant CP4-EPSPS gene as a selection marker was developed. Notably, all of the plants that were identified by PCR as transformants showed significant herbicide resistance. Additionally, this transformation protocol also highlighted: (i) the high yield of transgenic lines from calli (each gram of calli generated six transgenic lines); (ii) improved selection; and (iii) a higher transformation efficiency. This protocol provides a reliable tool for a routine procedure for the generation of resilient sugarcane plants.
PubMed: 38592870
DOI: 10.3390/plants13060852