Comprehensive analysis of the GALACTINOL SYNTHASE (GolS) gene family in citrus and the function of CsGolS6 in stress tolerance.

Galactinol synthase (GolS) catalyzes the first and rate-limiting step in the synthesis of raffinose family of oligosaccharides (RFOs), which serve as storage and transport sugars, signal transducers, compatible solutes and antioxidants in higher plants. The present work aimed to assess the potential functions of citrus GolS in mechanisms of stress response and tolerance. By homology searches, eight GolS genes were found in the genomes of Citrus sinensis and C. clementina. Phylogenetic analysis showed that there is a GolS ortholog in C. clementina for each C. sinensis GolS, which have evolved differently from those of Arabidopsis thaliana. Transcriptional analysis indicated that most C. sinensis GolS (CsGolS) genes show a low-level tissue-specific and stress-inducible expression in response to drought and salt stress treatments, as well as to 'Candidatus Liberibacter asiaticus' infection. CsGolS6 overexpression resulted in improved tobacco tolerance to drought and salt stresses, contributing to an increased mesophyll cell expansion, photosynthesis and plant growth. Primary metabolite profiling revealed no significant changes in endogenous galactinol, but different extents of reduction of raffinose in the transgenic plants. On the other hand, a significant increase in the levels of metabolites with antioxidant properties, such as ascorbate, dehydroascorbate, alfa-tocopherol and spermidine, was observed in the transgenic plants. These results bring evidence that CsGolS6 is a potential candidate for improving stress tolerance in citrus and other plants.

Genome-wide characterization and expression analysis of citrus NUCLEAR FACTOR-Y (NF-Y) transcription factors identified a novel NF-YA gene involved in drought-stress response and tolerance.

Nuclear factor Y (NF-Y) is a ubiquitous transcription factor found in eukaryotes. It is composed of three distinct subunits called NF-YA, NF-YB and NF-YC. NF-Ys have been identified as key regulators of multiple pathways in the control of development and tolerance to biotic and abiotic factors. The present study aimed to identify and characterize the complete repertoire of genes coding for NF-Y in citrus, as well as to perform the functional characterization of one of its members, namely CsNFYA5, in transgenic tobacco plants. A total of 22 genes coding for NF-Y were identified in the genomes of sweet orange (Citrus sinensis) and Clementine mandarin (C. clementina), including six CsNF-YAs, 11 CsNF-YBs and five CsNF-YCs. Phylogenetic analyses showed that there is a NF-Y orthologous in the Clementine genome for each sweet orange NF-Y gene; this was not observed when compared to Arabidopsis thaliana. CsNF-Y proteins shared the same conserved domains with their orthologous proteins in other organisms, including mouse. Analysis of gene expression by RNA-seq and EST data demonstrated that CsNF-Ys have a tissue-specific and stress inducible expression profile. qRT-PCR analysis revealed that CsNF-YA5 exhibits differential expression in response to water deficit in leaves and roots of citrus plants. Overexpression of CsNF-YA5 in transgenic tobacco plants contributed to the reduction of H2O2 production under dehydration conditions and increased plant growth and photosynthetic rate under normal conditions and drought stress. These biochemical and physiological responses to drought stress promoted by CsNF-YA5 may confer a productivity advantage in environments with frequent short-term soil water deficit.

Expression of the citrus CsTIP2;1 gene improves tobacco plant growth, antioxidant capacity and physiological adaptation under stress conditions.

MAIN CONCLUSION: Overexpression of the citrus CsTIP2;1 improves plant growth and tolerance to salt and drought stresses by enhancing cell expansion, H 2 O 2 detoxification and stomatal conductance. Tonoplast intrinsic proteins (TIPs) are a subfamily of aquaporins, belonging to the major intrinsic protein family. In a previous study, we have shown that a citrus TIP isoform, CsTIP2;1, is highly expressed in leaves and also transcriptionally regulated in leaves and roots by salt and drought stresses and infection by 'Candidatus Liberibacter asiaticus', the causal agent of the Huanglongbing disease, suggesting its involvement in the regulation of the flow of water and nutrients required during both normal growth and stress conditions. Here, we show that the overexpression of CsTIP2;1 in transgenic tobacco increases plant growth under optimal and water- and salt-stress conditions and also significantly improves the leaf water and oxidative status, photosynthetic capacity, transpiration rate and water use efficiency of plants subjected to a progressive soil drying. These results correlated with the enhanced mesophyll cell expansion, midrib aquiferous parenchyma abundance, H2O2 detoxification and stomatal conductance observed in the transgenic plants. Taken together, our results indicate that CsTIP2;1 plays an active role in regulating the water and oxidative status required for plant growth and adaptation to stressful environmental conditions and may be potentially useful for engineering stress tolerance in citrus and other crop plants.