ABA increased during salt stress triggers binding of AREB/ABFs to DcPSY2 promoter, boosting carotenoid and ABA levels as a positive feedback mechanism

Self-fertilization, which results in reduced fitness of offspring, is a common problem in hermaphrodite angiosperms. To prevent this, many plants utilize SI (self-incompatibility), which is determined by the multi-allelic S-locus, that allows discrimination between self (incompatible) and non-self (compatible) pollen by the pistil. In poppy (Papaver rhoeas), the pistil S-determinant (PrsS) is a small secreted protein which interacts with the pollen S-determinant PrpS, a ~20 kDa novel transmembrane protein. Interaction of matching pollen and pistil S-determinants results in self-recognition, initiating a Ca²⁺-dependent signalling network in incompatible pollen. This triggers several downstream events, including alterations to the cytoskeleton, phosphorylation of sPPases (soluble inorganic pyrophosphatases) and an MAPK (mitogen-activated protein kinase), increases in ROS (reactive oxygen species) and nitric oxide (NO), and activation of several caspase-like activities. This results in the inhibition of pollen tube growth, prevention of self-fertilization and ultimately PCD (programmed cell death) in incompatible pollen. The present review focuses on our current understanding of the integration of these signals with their targets in the SI/PCD network. We also discuss our recent functional expression of PrpS in Arabidopsis thaliana pollen.

Apple (Malus domestica) fruits accumulate negligible levels of carotenoids, antioxidant pigments that are precursors for vitamin A in humans. As vitamin A deficiency is an important public health issue, we aimed at increasing carotenoids in apple by constitutively expressing the Arabidopsis thaliana DXR gene, one of the key regulatory steps in the plastidial isoprenoid pathway. For this purpose, we optimized an Agrobacterium‐mediated transformation method in the commercial Fuji Raku Raku variety. This resulted in a shoot establishment efficiency of 0.75% at 20 weeks after infection. Molecular and microscopical analyses revealed that 80% of the hygromycin resistant shoots contained and expressed AtDXR:eGFP and that the AtDXR:eGFP fusion protein located in plastids. Transgenic seedlings displayed up to 3‐fold increase in total carotenoids and in individual carotenoids compared to the WT, correlating with an increased transcript abundance of endogenous carotenogenic genes such as MdDXS, MdPSY1, MdPSY2, MdPSY3, MdLCYB1, and MdLCYB2. In addition, buds of 2‐year‐old transgenic dormant trees showed an increment up to 3‐fold in lutein, and transient transformation of fruits revealed that AtDXR induced a 2‐fold increment in total carotenoids. Thus, these results suggest that DXR may be a good candidate for increasing carotenoid levels in apple fruits through metabolic engineering.

Carotenoids are pigments with important nutritional value in the human diet. As antioxidant molecules, they act as scavengers of free radicals enhancing immunity and preventing cancer and cardiovascular diseases. Moreover, α-carotene and β-carotene, the main carotenoids of carrots (Daucus carota) are precursors of vitamin A, whose deficiency in the diet can trigger night blindness and macular degeneration. With the aim of increasing the carotenoid content in fruit flesh, three key genes of the carotenoid pathway, phytoene synthase (DcPSY2) and lycopene cyclase (DcLCYB1) from carrots, and carotene desaturase (XdCrtI) from the yeast Xanthophyllomyces dendrorhous, were optimized for expression in apple and cloned under the Solanum chilense (tomatillo) polygalacturonase (PG) fruit specific promoter. A biotechnological platform was generated and functionally tested by subcellular localization, and single, double and triple combinations were both stably transformed in tomatoes (Solanum lycopersicum var. Microtom) and transiently transformed in Fuji apple fruit flesh (Malus domestica). We demonstrated the functionality of the S. chilense PG promoter by directing the expression of the transgenes specifically to fruits. Transgenic tomato fruits expressing DcPSY2, DcLCYB1, and DcPSY2-XdCRTI, produced 1.34, 2.0, and 1.99-fold more total carotenoids than wild-type fruits, respectively. Furthermore, transgenic tomatoes expressing DcLCYB1, DcPSY2-XdCRTI, and DcPSY2-XdCRTI-DcLCYB1 exhibited an increment in β-carotene levels of 2.5, 3.0, and 2.57-fold in comparison with wild-type fruits, respectively. Additionally, Fuji apple flesh agroinfiltrated with DcPSY2 and DcLCYB1 constructs showed a significant increase of 2.75 and 3.11-fold in total carotenoids and 5.11 and 5.84-fold in β-carotene, respectively whereas the expression of DcPSY2-XdCRTI and DcPSY2-XdCRTI-DcLCYB1 generated lower, but significant changes in the carotenoid profile of infiltrated apple flesh. The results in apple demonstrate that DcPSY2 and DcLCYB1 are suitable biotechnological genes to increase the carotenoid content in fruits of species with reduced amounts of these pigments.

No abstract