Citrus NIP5;1 aquaporin regulates cell membrane water permeability and alters PIPs plasma membrane localization.

KEY MESSAGE: The ER or donut-like structures localized aquaporin NIP5;1, which interacts with PIPs and alters their localization from plasma membrane to donut-like structures, regulates water permeability. NOD26-like intrinsic proteins (NIPs) play important roles in nutrient uptake and response to various stresses. However, there have been few studies of their functions in water transportation in citrus. Here, we demonstrate the functions of a novel citrus NIP aquaporin (CsNIP5;1) via multiple physiological and biochemical experiments. CsNIP5;1 showed high water permeability when expressed in Xenopus laevis oocytes and yeast. However, subcellular localization assays showed that this protein was localized in the endoplasmic reticulum (ER) or donut-like structures in citrus callus and tobacco leaf. Meanwhile, overexpression of CsNIP5;1 led to a reduction in the water permeability of citrus callus. Protein-protein interaction experiments and subcellular localization assays further revealed that CsNIP5;1 physically interacted with PIPs (CsPIP1;1 and AtPIP2;1), which altered their subcellular localization from the plasma membrane to donut-like structures. Together, CsNIP5;1 was identified as a good water channel when expressed in oocytes and yeast. Meanwhile, CsNIP5;1 participated in the regulation of water permeability of citrus callus, which may be associated with CsNIP5;1-induced re-localization of water channels PIPs. In summary, these results provide new insights into the regulatory mechanism of AQPs-mediated water diffusion.

Variations of membrane fatty acids and epicuticular wax metabolism in response to oleocellosis in lemon fruit.

Oleocellosis is a physiological disorder causing blemishes on fruit surface. This study investigated the influence of oleocellosis on the membrane fatty acids and wax in lemon fruit rinds at the morphological, physiological, metabolic and molecular levels by using a variety with a high incidence rate of oleocellosis (green lemon). Oleocellosis-damaged rinds showed loose and flaky wax layers with more fissures on the surface, as well as higher contents of C16 and C18 fatty acids and very long chain (VLC) fatty alkanes while lower contents of VLC fatty aldehydes. The main differentially expressed genes, including FabZ, FAD2 and SAD6 involved in the accumulation of C16 and C18 fatty acids and CER1 involved in the transformation of VLC fatty aldehydes to VLC fatty alkanes, were up-regulated by oleocellosis. These results indicate that oleocellosis accelerates the accumulation of membrane free fatty acids and transformation of VLC fatty aldehydes to VLC fatty alkanes.

An R2R3-MYB transcription factor represses the transformation of α- and ß-branch carotenoids by negatively regulating expression of CrBCH2 and CrNCED5 in flavedo of Citrus reticulate.

Although the functions of carotenogenic genes are well documented, little is known about the mechanisms that regulate their expression, especially those genes involved in α - and ß-branch carotenoid metabolism. In this study, an R2R3-MYB transcriptional factor (CrMYB68) that directly regulates the transformation of α- and ß-branch carotenoids was identified using Green Ougan (MT), a stay-green mutant of Citrus reticulata cv Suavissima. A comprehensive analysis of developing and harvested fruits indicated that reduced expression of ß-carotene hydroxylases 2 (CrBCH2) and 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5) was responsible for the delay in the transformation of α- and ß-carotene and the biosynthesis of ABA. Additionally, the expression of these genes was negatively correlated with the expression of CrMYB68 in MT. Further, electrophoretic mobility shift assays (EMSAs) and dual luciferase assays indicated that CrMYB68 can directly and negatively regulate CrBCH2 and CrNCED5. Moreover, transient overexpression experiments using leaves of Nicotiana benthamiana indicated that CrMYB68 can also negatively regulate NbBCH2 and NbNCED5. To overcome the difficulty of transgenic validation, we quantified the concentrations of carotenoids and ABA, and gene expression in a revertant of MT. The results of these experiments provide more evidence that CrMYB68 is an important regulator of carotenoid metabolism.