No abstract

The binding protein BiP is an endoplasmic reticulum (ER)-resident member of the HSP70 stress-related protein family, which is essential for the constitutive function of the ER. In addition to responding to a variety of environmental stimuli, plant BiP exhibits a tissue-specific regulation. We have isolated two soybean BiP genomic clones, designated gsBiP6 and gsBiP9, and different extensions of their 5' flanking sequences were fused to beta-glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Transgenic plants displayed prominent GUS activity in the vascular bundles of roots and shoots as well as in regions of intense cell division, such as procambial region and apical meristems. Promoter deletion analyses identified two cis-regulatory functional domains that are important for the spatially-regulated activation of BiP expression under normal plant development. While an AT-rich enhancer-like sequence, designated cis-acting regulatory domain 1, CRD1 (-358 to -211, on gsBiP6), activated expression of the BiP minimal promoter in all organs analyzed, BiP promoter activity in meristematic tissues and phloem cells required the presence of a second activating domain, CRD2 (-211 to -80). Apparently, the CRD2 sequence also harbors negative cis-acting elements, because removal of this region caused activation of gsBiP6 promoter in parenchymatic xylem rays. These results suggest that the tissue-specific control of BiP gene expression requires a complex integration of multiple cis-acting regulatory elements on the promoter.

The employment of biotechnology-based approaches such as somatic embryogenesis has been applied to several plants including Coffea sp. Despite the economic importance of this genus, few information about the role of key regulatory genes in somatic embryogenesis in coffee is available. This work provides information about ABI3 and VAL2 genes performance by RT-qPCR during indirect somatic embryogenesis of Coffea arabica. To achieve this, bioinformatics analysis was performed to identify the genes of the B3 superfamily in the coffee genome. The cell suspensions lines presented similar histological and regeneration patterns, yielding of up to 6.6 embryos per 1 mg of embryogenic aggregates at 7 months. We have identified possible orthologs for VAL2 (Cc06g00410) and ABI3 (Cc01g17380) as well as the other members belonging to superfamily B3. The CaABI3 expression was higher in dedifferentiated competent cells for somatic embryogenesis as compared with non-embryogenic calli. Whereas the expression of VAL2 gene is more active in cotyledonary embryos and plantlets, showing its clear performance in the embryogenesis late stages. The present study suggests that CaABI3 gene could be potentially used as a biomarker for embryogenic process improvement. The good plantlets development obtained from the protocol used may be a reflection of the high expression of CaVAL2 in cotyledonary embryos and plantlets. Key message The activity of CaABI3 is correlated to embryogenic potential with highly expressed in embryogenic masses and expression of the VAL2 gene is increased at the end of the embryogenic process.

In contrast to yeast or mammalian counterpart, BiP (Binding Protein) from several plant species, such as maize, tobacco, Arabidopsis and soybean, is encoded by a multigene family. A systematic characterization and analysis of soybean BiP expression have provided evidence for the existence of multiple, complex regulatory mechanisms controlling plant BiP gene expression. In support of this observation, the soybean BiP gene family has been shown to exhibit organ-specific expression and differential regulation in response to abiotic stresses through distinct signaling pathways. As a member of the stress-regulated HSP70 family of protein, the elucidation of plant BiP function and regulation is likely to lead do new strategies to enhance crop tolerance to environmental stress. Consistent with this observation, transgenic plants overexpressing soybean BiP have demonstrated to exhibit increased tolerance to ER (endoplasmic reticulum) stressors during seed germination and enhanced tolerance to water deficit during plant growth.

Coffea canephora (2n = 2x = 22 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 β-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.