Abscisic Acid Deficiency Alters Epicuticular Wax Metabolism and Morphology That Leads to Increased Cuticle Permeability During Sweet Orange (Citrus sinensis) Fruit Ripening

Abstract

Citrus fruit ripening is coupled with the synthesis and deposition of epicuticular waxes, which reduces water loss during fruit postharvest storage. Although abscisic acid (ABA) is a major regulator of citrus fruit ripening, whether ABA mediates epicuticular wax formation during this process remains poorly understood. We investigated the implication of ABA in cuticle properties and epicuticular wax metabolism, composition, and morphology by comparing the Navelate orange [Citrus sinensis (L.) Osbeck] and its ABA biosynthesis-impaired mutant Pinalate in four ripening stages. ABA deficiency had minor effects on cuticle thickness and epicuticular wax load, but correlated with cuticle permeability. ABA content aligned with mostly fatty acids accumulation in both cultivars, and also with specific alkane, terpenoid, and aldehyde constituents in the parental fruit. In turn, cuticle permeability correlated with the fatty acid profile during fruit ripening in the Navelate and Pinalate, and with primary alcohols, terpenoids, and aldehydes, but only in the mutant fruit. Low ABA levels increased the susceptibility of waxes to crack and were lost from the epicuticular layer. The RNA-seq analysis highlighted the differential regulation of a list of 87 cuticle-related genes between genotypes and ripening stages. Changes in the gene expression of the selected genes in both cultivars were consistent with the content of the aliphatics and terpenoid fractions during ripening. The results suggest a role for ABA in the regulation of fatty acid content and primary alcohol composition, and point out the importance of alkane and triterpenoid for controlling water permeance through fruit cuticles.

Albedo- and Flavedo-Specific Transcriptome Profiling Related to Penicillium digitatum Infection in Citrus Fruit

“…Total RNA was extracted from 1 g of the flavedo or albedo samples, previously frozen, homogenized, and kept at −80 °C, as previously described [ 23 ]. The RNA concentration was determined by a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and its integrity was verified by migrating 1 µg of RNA on agarose gel [ 23 ].…”

“…Total RNA was extracted from 1 g of the flavedo or albedo samples, previously frozen, homogenized, and kept at −80 °C, as previously described [ 23 ]. The RNA concentration was determined by a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and its integrity was verified by migrating 1 µg of RNA on agarose gel [ 23 ]. Before the RNA-Seq analysis, RNA quality was assessed by the Agilent 2100 Total RNA Bioanalyzer (Agilent Technologies, Madrid, Spain) using the RNA 6000 Nano Kit (Agilent Technologies, Madrid, Spain).…”

“…Three biological replicates of 2 μg of the RNA of the albedo and flavedo samples from the freshly harvested Navelate oranges, and from the flavedo and albedo samples collected at 1 dpi from the fruit inoculated with P. digitatum and their respective mock-wounded control fruit, were employed to construct sequencing libraries, as previously described [ 23 ]. They were constructed by means of the TruSeq Stranded mRNA Library Prep Kit ® with PolyA selection for ribosomal RNA depletion (Illumina, San Diego, CA, USA) following the manufacturer’s recommendations.…”

“…For the gene expression analysis, total RNA was treated with ribonuclease-free DNase (Thermo Fisher Scientific, Wilmington, DE, USA) following the manufacturer’s instructions to remove genomic DNA contaminations. A RT-qPCR analysis was carried out following the procedure described in [ 23 ] to validate the RNA-Seq results, and to examine the expression pattern of the genes selected in the flavedo and albedo disc samples taken from the mock-wounded (control) fruit and from the fruit inoculated with P. digitatum . The cDNA from each sample was synthesized from 2 μg of total RNA by SuperScript III RT (Thermo Fisher Scientific, Wilmington, DE, USA) and ribonuclease inhibitor (Thermo Fisher Scientific, Wilmington, DE, USA), as indicated by the manufacturer.…”

The Combination of Abscisic Acid (ABA) and Water Stress Regulates the Epicuticular Wax Metabolism and Cuticle Properties of Detached Citrus Fruit

“…In fruit, the participation of ABA in fruit water stress regulation has also been demonstrated in both climacteric and non-climacteric fruit [ 4 ]. More recently, a relationship between ABA and cuticle, the first barrier to limit water loss, has been suggested in model plants [ 5 , 6 ] and during fruit ripening [ 7 , 8 , 9 , 10 ]. ABA treatment increased wax load in Arabidopsis plants, mainly due to changes in the alkane fraction [ 5 ].…”