An improved protocol for Agrobacteriummediated transformation of Duncan grapefruit (Citrus paradisi Macf.) epicotyl explants was developed by examining the effects of six different factors on the efficiency of transformation and combining the best treatment for each factor. The preculturing of explants and the composition of the cocultivation medium were the factors that most influenced transformation efficiency. The optimized protocol was successfully employed in the production of transgenic grapefruit plants containing the carotenoid biosynthetic genes phytoene synthase, phytoene desaturase, or lycopene-β-cyclase under constitutive expression. With an eventual goal of metabolically engineering grapefruit with multiple genes, hygromycin as a selectable marker and BIBAC as a transformation vector for large pieces of DNA were also tested.

Late Embryogenesis Abundant (LEA) proteins are an ubiquitous group of polypeptides that were first described to accumulate during plant seed dehydration, at the later stages of embryogenesis. Since then they have also been recorded in vegetative plant tissues experiencing water limitation and in anhydrobiotic bacteria and invertebrates and, thereby, correlated with the acquisition of desiccation tolerance. This study provides the first comprehensive study about the LEA gene family in sweet orange (Citrus sinensis L. Osb.), the most important and widely grown fruit crop around the world. A surprisingly high number (72) of genes encoding C. sinensis LEAs (CsLEAs) were identified and classified into seven groups (LEA_1, LEA_2, LEA_3 and LEA_4, LEA_5, DEHYDRIN and SMP) based on their predicted amino acid sequences and also on their phylogenetic relationships with the complete set of Arabidopsis thaliana LEA proteins (AtLEAs). Approximately 60% of the CsLEAs identified in this study belongs to the unusual LEA_2 group of more hydrophobic LEA proteins, while the other LEA groups contained a relatively small number of members typically hydrophilic. A correlation between gene structure and motif composition was observed within each LEA group. Investigation of their chromosomal localizations revealed that the CsLEAs were non-randomly distributed across all nine chromosomes and that 33% of all CsLEAs are segmentally or tandemly duplicated genes. Analysis of the upstream sequences required for transcription revealed the presence of various stress-responsive cis-acting regulatory elements in the promoter regions of CsLEAs, including ABRE, DRE/CRT, MYBS and LTRE. Expression analysis using both RNA-seq data and quantitative real-time RT-PCR (qPCR) revealed that the CsLEA genes are widely expressed in various tissues, and that many genes containing the ABRE promoter sequence are induced by drought, salt and PEG. These results provide a useful reference for further exploration of the CsLEAs functions and applications on crop improvement.

An improved method for the Agrobacterium infiltration of epicotyl segments of 'Pineapple' sweet orange [Citrus sinensis (L.) Osbeck] and 'Swingle' citrumelo [Citrus paradisi Macf. X Poncirus trifoliata (L.) Raf.] was developed in order to increase transformation frequency. Sonication-assisted Agrobacterium-mediated transformation (SAAT), vacuum infiltration, and a combination of the two procedures were compared with conventional Agrobacterium-mediated inoculation method ('dipping' method). It was observed that the morphogenic potential of the epicotyl segments decreased as the duration of SAAT and vacuum treatments increased. Transient GUS expression was not affected by the different SAAT treatments, but it was significantly enhanced by the vacuum infiltration treatments. The highest transformation efficiencies were obtained when the explants were subjected to a combination of SAAT for 2 s followed by 10 min of vacuum infiltration. PCR and Southern blot analysis of the uidA gene were used to confirm the integration of the transgenes. The transformation frequencies achieved in this study (8.4% for 'Pineapple' sweet orange and 11.2% for 'Swingle' citrumelo) are the highest ones reported for both cultivars.

Somatic embryogenesis is a morphogenetic event where somatic cells have the ability to produce embryos without gamete fusion. It is used as a technique for plant mass propagation. It is a process that has six well defined steps such as induction, expression, development, maturation, germination and plant conversion. These steps are characterized by distinct physiological, morphological and molecular events. Although somatic embryogenesis has been established in several plant species, there remains many problems to be solved. The main problem in somatic embryogenesis is the large number of abnormal embryos produced which cannot germinate nor convert into normal plants. Abnormalities in somatic embryos (SE) can be generated by genetic or epigenetic changes in the DNA. These changes in the DNA can be influenced by external factors such as the use of plant growth regulators and mutagenic substances or stress factors applied to the plant tissue such as high and low temperatures, drought, salinity, and heavy metals. Abnormalities generated by genetic changes in the DNA are hardly reversible; however, abnormalities generated by epigenetic changes may be reversible and the abnormal embryos are able to produce normal plants in most cases. This review focuses on the identification of the main factors that can cause abnormal SE development in different plant species, suggest how SE abnormalities are related to somaclonal variations and identify which genes may be involved with embryo abnormalities. Zygotic embryo abnormalities in Arabidopsis thaliana mutants are listed with the aim to understand the main genetic mechanisms involved in embryo aberrations. Key messageThe abnormalities in somatic embryos are related to the use of 2,4-D in most of the published protocols, this sintetic auxin disrupts the endogenous auxin balance and the auxin polar transportation interfering with the embryo apical-basal polarity.

No abstract

Altered protein phosphorylation states of several proteins are closely associated with Alzheimer's disease (AD). Among these are the amyloid-β protein precursor (AβPP) and the tau protein. In fact, altered protein phosphorylation states already provide strong biomarkers for AD diagnosis, as is the case with hyperphosphorylated tau. It follows that modulating signaling cascades provides an attractive avenue for exploring novel therapeutic strategies. This review focuses on some of the major protein kinases and protein phosphatases relevant to AD. Of particular relevance, posttranslational modifications dynamically regulate protein activity, subcellular localization, and stability. Protein phosphorylation states can mediate complex formation as well as regulate protein function, and this is important for cellular physiology but can likewise contribute to the development of neuropathological conditions. Furthermore, applying a system approach provides a more comprehensive understanding of the signaling events associated with AD and highlights possible convergence points that may contribute to the different AD pathological hallmarks.

The NAC (NAM, ATAF1, -2, and CUC2) gene family encodes a large family of plant-specific transcription factors that play diverse roles in plant development and stress regulation. In this study, we performed a survey of citrus NAC transcription factors in the HarvEST: Citrus database, in which 45 NAC domain-containing proteins were identified and phylogenetically classified into 13 different subfamilies. The results suggest the existence of a structurally diversified family of NAC transcription factors in citrus, which has not been previously characterized. One of these NAC genes, CsNAC1 was found to be a member of the stress-NAC subfamily, whose homologs from other plant species function in pathways of environmental stress response and tolerance, and was further characterized. The CsNAC1 deduced protein was shown to contain the five N-terminal A through E NAC subdomains, a C-terminal region containing three transcriptional activation motifs, and a predicted NAC nuclear localization signal, consistent with its putative role as a NAC transcription factor. In silico analysis indicated that CsNAC1 was primarily expressed in leaves and shoot meristems, and was involved in general stress responses. Quantitative real-time reverse transcription PCR analysis revealed that CsNAC1 was strongly induced by drought stress in leaves of Citrus reshni and Citrus limonia, and also by salt stress, cold, and ABA in leaves and roots of C. reshni. Collectively, these results suggest that CsNAC1 encodes a novel stress-responsive NAC transcription factor that is potentially useful for engineering tolerance to multiple abiotic stresses in citrus.

Abscisic acid is a plant hormone that participates in essential plant physiological processes, especially during adaptation to many environmental stresses, such as water deficit. The relationship between ABA accumulation and the expression of putative carotenoid cleavage dioxygenase (CCD) genes was investigated in the pot-cultivated leaves and roots of the 'Rangpur' lime and 'Sunki Maravilha' mandarin plants. Transpiration, stomatal resistance and leaf growth were evaluated when these genotypes were subjected to continuous water deficit. Under water deficit conditions, the 'Rangpur' lime extracts used greater amounts of water when compared to the 'Sunki Maravilha' plants, which reached the greatest stomatal resistance 5 days before 'Rangpur' lime. When subjected to water deficit, the roots and leaves of 'Sunki Maravilha' showed a progressive increase in ABA accumulation; however, in 'Rangpur' lime, alternations between high and low ABA concentrations were observed. These results suggest a retroactive feeding regulation by ABA. In 'Rangpur' lime the NCED2, NCED3 and CCD4a genes were expressed at the highest levels in the roots, and NCED5 was highly expressed in the leaves; in 'Sunki Maravilha', the NCED2 and NCED5 genes were most highly expressed in the roots, and NCED2 was most highly expressed in the leaves. However, for both genotypes, the transcription of these genes only correlated with ABA accumulation during the most severe water deficit conditions. The 'Rangpur' lime behaved as a vigorous rootstock; the leaf growth remained unaltered even when water was scarce. However, 'Sunki Maravilha' adaptation was based on the equilibrium of the response between the root and the aerial tissues due to water restriction. The use of the Sunki mandarin in combination with a scion with similar characteristics as its own, which responds to water deficit stress by accumulating ABA in the leaves, may display good drought tolerance under field conditions.