Differential fine-tuning of gene expression regulation in coffee leaves by CcDREB1D promoter haplotypes under water deficit.

Despite the importance of the DREB1D gene (also known as CBF4) in plant responses to water deficit and cold stress, studies analysing its regulation by transgenic approaches are lacking. In the current work, a functional study of three CcDREB1D promoter haplotypes (named HP15, HP16 and HP17) isolated from drought-tolerant and droughtsensitive clones of Coffea canephora was carried out in plants of C. arabica stably transformed by Agrobacterium tumefaciens by analysing their ability to regulate the expression of the uidA reporter gene in response to water deficit mimicked by polyethylene glycol (−2.0 MPa) and low relative humidity treatments

Multiplex CRISPR/Cas9-mediated knockout of the phytoene desaturase gene in Coffea canéfora.

Coffea canephora (2n=2x22 chromosomes) is a species with extensive genetic diversity and desirable agronomic traits for coffee breeding programs. However, obtaining a new coffee cultivar through conventional breeding techniques may require more than 30 years of crossing cycles and selection, which hampers the effort of keeping up with market demands and rapidly proposing more resilient to climate change varieties. Although, the application of modern biotechnology tools such as precision genetic engineering technologies may enable a faster cultivar development process. Therefore, we aimed to validate the CRISPR/Cas9 system to generate mutations on a selected genotype of C. canephora, the clone 14. Embryogenic calli and a multiplex binary vector containing two sgRNAs targeting different exons of the CcPDS gene were used. The sgRNAs were under the C. canephora U6 promoter regulation. The target gene encodes phytoene desaturase, an enzyme essential for photosynthesis involved in B-carotene biosynthesis. Somatic seedlings and embryos with albino, variegated and green phenotypes regenerated after Agrobacterium tumefaciens-mediated genetic transformation were analyzed by verifying the insertion of the Cas9 gene and later by sequencing the sgRNAs target regions in the genome of Robusta modified seedlings. Among them, 77% had the expected mutations, and of which, 50% of them had at least one target with a homozygous mutation. The genotype, temperature of co-cultivation with the bacteria, and light intensity used for subsequent embryo regeneration appeared to strongly influence the successful regeneration of plants with a mutated CcPDS gene in the Coffea genus

Regioselective Synthesis of Benzimidazolones via Cascade C–N Coupling of Monosubstituted Ureas

A direct method for the regioselective construction of benzimidazolones is reported wherein a single palladium catalyst is employed to couple monosubstituted urea substrates with differentially substituted 1,2-dihaloaromatic systems. In this method, the catalyst is able to promote a cascade of two discrete chemoselective C–N bond-forming processes that allows the highly selective and predictable formation of complex heterocycles from simple, readily available starting materials.National Institutes of Health (U.S.) (Award GM58160)National Institutes of Health (U.S.) (Award GM099817)Lanxess CorporationMassachusetts Institute of Technology. Undergraduate Research Opportunities Progra

Cisgenesis and intragenesis as new strategies for crop improvement

Cisgenesis and intragenesis are emerging plant breeding technologies which offer great promise for future acceptance of genetically engineered crops. The techniques employ traditional genetic engineering methods but are confined to transferring of genes and genetic elements between sexually compatible species that can breed naturally. One of the main requirements is the absence of selectable marker genes (such as antibiotic resistance genes) in the genome. Hence the sensitive issues with regard to transfer of foreign genes and antibiotic resistance are overcome. It is a targeted technique involving specific locus; therefore, linkage drag that prolongs the time for crop improvement in traditional breeding does not occur. It has great potential for crop improvement using superior alleles that exist in the untapped germplasm or wild species. Cisgenic and intragenic plants may not face the same stringent regulatory assessment for field release as transgenic plants which is a clear added advantage that would save time. In this chapter, the concepts of cis/intragenesis and the prerequisites for the development of cis/intragenesis plants are elaborated. Strategies for marker gene removal after selection of transformants are discussed based on the few recent reports from various plant species

Molecular genetics of rice root development

Plant roots have a large range of functions, including acquisition of water and nutrients, as well as structural support. Dissecting the genetic and molecular mechanisms controlling rice root development is critical for the development of new rice ideotypes that are better adapted to adverse conditions and for the production of sustainably achieved rice yield potential. Most knowledge regarding the gene networks involved in root development has been accumulated in the model dicotyledon plant species Arabidopsis thaliana. Rice, the model monocotyledon species, presents several singularities compared to A. thaliana, including a root architecture characterized by a fibrous root system comprising five types of embryonic and postembryonic roots. The anatomy and morphology of the rice root system, which is typical for a cereal, differs from that of A. thaliana, for instance, by the presence of a lysigenous cortex and additional cell layers compared to the dicotyledon model. Moreover, the structure and functions of the root apical meristem (RAM) of rice are distinct from those of A. thaliana. Recently, several rice root mutants have been identified via forward or reverse genetics, and these will aid in forming hypothesis to characterize either the divergence or conservation of genetic pathways relative to A. thaliana. Furthermore, these mutants will help to identify key genes in rice roots that may be missing in A. thaliana. This review summarizes both classical and recent data concerning the molecular genetics of rice root development, including root anatomy and morphology, RAM structure, RAM patterning, and root mutant

Full moonlight-induced circadian clock entrainment in Coffea arabica

Background It is now well documented that moonlight affects the life cycle of invertebrates, birds, reptiles, and mammals. The lunisolar tide is also well-known to alter plant growth and development. However, although plants are known to be very photosensitive, few studies have been undertaken to explore the effect of moonlight on plant physiology. Results Here for the first time we report a massive transcriptional modification in Coffea arabica genes under full moonlight conditions, particularly at full moon zenith and 3 h later. Among the 3387 deregulated genes found in our study, the main core clock genes were affected. Conclusions Moonlight also negatively influenced many genes involved in photosynthesis, chlorophyll biosynthesis and chloroplast machinery at the end of the night, suggesting that the full moon has a negative effect on primary photosynthetic machinery at dawn. Moreover, full moonlight promotes the transcription of major rhythmic redox genes and many heat shock proteins, suggesting that moonlight is perceived as stress. We confirmed this huge impact of weak light (less than 6 lx) on the transcription of circadian clock genes in controlled conditions mimicking full moonlight