SummaryFlowering relies on signaling networks that integrate endogenous and external cues. Normally, plants ower at a particular season, re¯ecting day length and/or temperature cues. However, plants can surpass this seasonal regulation and show precocious¯owering under stress environmental conditions. Here, we show that UV-C light stress activates the transition to¯owering in Arabidopsis thaliana through salicylic acid (SA). Moreover, SA also regulates¯owering time in non-stressed plants, as SA-de®cient plants are latē owering. The regulation of¯owering time by SA seems to involve the photoperiod and autonomous pathways, but it does not require the function of the¯owering time genes CONSTANS (CO), FCA, or FLOWERING LOCUS C (FLC ).

SummaryOrange is a major crop and an important source of health-promoting bioactive compounds. Increasing the levels of specific antioxidants in orange fruit through metabolic engineering could strengthen the fruit's health benefits. In this work, we have afforded enhancing the b-carotene content of orange fruit through blocking by RNA interference the expression of an endogenous b-carotene hydroxylase gene (Csb-CHX) that is involved in the conversion of b-carotene into xanthophylls. Additionally, we have simultaneously overexpressed a key regulator gene of flowering transition, the FLOWERING LOCUS T from sweet orange (CsFT), in the transgenic juvenile plants, which allowed us to obtain fruit in an extremely short period of time. Silencing the Csb-CHX gene resulted in oranges with a deep yellow ('golden') phenotype and significant increases (up to 36-fold) in b-carotene content in the pulp. The capacity of b-carotene-enriched oranges for protection against oxidative stress in vivo was assessed using Caenorhabditis elegans as experimental animal model. Golden oranges induced a 20% higher antioxidant effect than the isogenic control. This is the first example of the successful metabolic engineering of the b-carotene content (or the content of any other phytonutrient) in oranges and demonstrates the potential of genetic engineering for the nutritional enhancement of fruit tree crops.

Citrus is the most important fruit tree crop in the world, with a production of more than 100 million tons annually. The area of origin of Citrus is believed to be southeastern Asia, where its domestication started. It has become clear that only citron, mandarin, and pummelo are true species within genus Citrus, being other important Citrus types, as sweet orange, sour orange, lemon, lime, grapefruit and other mandarins originated from hybridization between these ancestral species. In spite of the many efforts put in classical breeding programs in the last 100 years, current citrus industry relies on various groups of varieties that are grafted onto rootstocks adapted to different abiotic and biotic stresses. Most of these genotypes have been generated by chance, mostly as budsports but also as natural hybrids or seedlings selected by men in the wild or in orchards. Citrus breeding is complicated due to its complex reproductive biology. In this context, genetic transformation offers an important alternative for the genetic improvement of citrus. Moreover, it is probably the most efficient approach to make reverse genetics in citrus to investigate gene function and thus to gain better understanding in metabolic processes and plant‐pathogen‐environment interactions.

BackgroundThe future of genetic transformation as a tool for the improvement of fruit trees depends on the development of proper systems for the assessment of unintended effects in field-grown GM lines. In this study, we used eight transgenic lines of two different citrus types (sweet orange and citrange) transformed with the marker genes β-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) as model systems to study for the first time in citrus the long-term stability of transgene expression and whether transgene-derived pleiotropic effects occur with regard to the morphology, development and fruit quality of orchard-grown GM citrus trees.ResultsThe stability of the integration and expression of the transgenes was confirmed in 7-year-old, orchard-grown transgenic lines by Southern blot analysis and enzymatic assays (GUS and ELISA NPTII), respectively. Little seasonal variation was detected in the expression levels between plants of the same transgenic line in different organs and over the 3 years of analysis, confirming the absence of rearrangements and/or silencing of the transgenes after transferring the plants to field conditions. Comparisons between the GM citrus lines with their non-GM counterparts across the study years showed that the expression of these transgenes did not cause alterations of the main phenotypic and agronomic plant and fruit characteristics. However, when comparisons were performed between diploid and tetraploid transgenic citrange trees and/or between juvenile and mature transgenic sweet orange trees, significant and consistent differences were detected, indicating that factors other than their transgenic nature induced a much higher phenotypic variability.ConclusionsOur results indicate that transgene expression in GM citrus remains stable during long-term agricultural cultivation, without causing unexpected effects on crop characteristics. This study also shows that the transgenic citrus trees expressing the selectable marker genes that are most commonly used in citrus transformation were substantially equivalent to the non-transformed controls with regard to their overall agronomic performance, as based on the use of robust and powerful assessment techniques. Therefore, future studies of the possible pleiotropic effects induced by the integration and expression of transgenes in field-grown GM citrus may focus on the newly inserted trait(s) of biotechnological interest.

Background/ObjectiveDespite potential benefits granted by genetically modified (GM) fruit trees, their release and commercialization raises concerns about their potential environmental impact, and the transfer via pollen of transgenes to cross-compatible cultivars is deemed to be the greatest source for environmental exposure. Information compiled from field trials on GM trees is essential to propose measures to minimize the transgene dispersal. We have conducted a field trial of seven consecutive years to investigate the maximum frequency of pollen-mediated crop-to-crop transgene flow in a citrus orchard, and its relation to the genetic, phenological and environmental factors involved.Methodology/Principal FindingsThree different citrus genotypes carrying the uidA (GUS) tracer marker gene (pollen donors) and a non-GM self-incompatible contiguous citrus genotype (recipient) were used in conditions allowing natural entomophilous pollination to occur. The examination of 603 to 2990 seeds per year showed unexpectedly low frequencies (0.17–2.86%) of transgene flow. Paternity analyses of the progeny of subsets of recipient plants using 10 microsatellite (SSR) loci demonstrated a higher mating competence of trees from another non-GM pollen source population that greatly limited the mating chance of the contiguous cross-compatible and flowering-synchronized transgenic pollen source. This mating superiority could be explained by a much higher pollen competition capacity of the non-GM genotypes, as was confirmed through mixed-hand pollinations.Conclusions/SignificancePollen competition strongly contributed to transgene confinement. Based on this finding, suitable isolation measures are proposed for the first time to prevent transgene outflow between contiguous plantings of citrus types that may be extendible to other entomophilous transgenic fruit tree species.