An Intracellular Laccase Is Responsible for Epicatechin-Mediated Anthocyanin Degradation in Litchi Fruit Pericarp.

In contrast to the detailed molecular knowledge available on anthocyanin synthesis, little is known about its catabolism in plants. Litchi (Litchi chinensis) fruit lose their attractive red color soon after harvest. The mechanism leading to quick degradation of anthocyanins in the pericarp is not well understood. An anthocyanin degradation enzyme (ADE) was purified to homogeneity by sequential column chromatography, using partially purified anthocyanins from litchi pericarp as a substrate. The purified ADE, of 116 kD by urea SDS-PAGE, was identified as a laccase (ADE/LAC). The full-length complementary DNA encoding ADE/LAC was obtained, and a polyclonal antibody raised against a deduced peptide of the gene recognized the ADE protein. The anthocyanin degradation function of the gene was confirmed by its transient expression in tobacco (Nicotiana benthamiana) leaves. The highest ADE/LAC transcript abundance was in the pericarp in comparison with other tissues, and was about 1,000-fold higher than the polyphenol oxidase gene in the pericarp. Epicatechin was found to be the favorable substrate for the ADE/LAC. The dependence of anthocyanin degradation by the enzyme on the presence of epicatechin suggests an ADE/LAC epicatechin-coupled oxidation model. This model was supported by a dramatic decrease in epicatechin content in the pericarp parallel to anthocyanin degradation. Immunogold labeling transmission electron microscopy suggested that ADE/LAC is located mainly in the vacuole, with essential phenolic substances. ADE/LAC vacuolar localization, high expression levels in the pericarp, and high epicatechin-dependent anthocyanin degradation support its central role in pigment breakdown during pericarp browning.

3-Hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMG1) is highly associated with the cell division during the early stage of fruit development which determines the final fruit size in Litchi chinensis.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34), an enzyme catalyzing the first committed step in the mevalonic acid (MVA) pathway for the biosynthesis of isoprenoids, has been reported to be involved in the fruit size determination through the regulation of early cell division. In litchi, the cell number achieved by this early cell division determines the final fruit size, but whether HMGR plays any role in this process was unknown. In this study, we set out to address this question with gene cloning and expression analysis in fruits of different pheno- or genotypes. We found that the litchi genome includes two HMGR homologues, denoted as LcHMG1 and LcHMG2. Despite 70% sequence identity at the amino acid level, they exhibited distinct expression patterns during litchi fruit development. LcHMG1 expression was highest in the early stage of fruit development, correlated with the high level of cell division. Absolute levels of LcHMG1 expression varied among fruits of different pheno- or genotypes, with expression in large-fruited types reaching higher levels for longer duration compared to that in small-fruited types. The expression patterns for LcHMG1 strongly suggest that this gene is involved in early cell division and fruit size determination in litchi. In contrast, LcHMG2 was most highly expressed in the late stage of fruit development, in association with biosynthesis of isoprenoid compounds required for later cell enlargement. These findings provided new insights on the function of HMGR genes during fruit development.

Distribution changes of calcium and programmed cell death in the pistil of litchi (Litchi chinensis Sonn.) flower during its development.

Potassium pyroantimonate precipitation method was used for investigating calcium distribution and cell ultrastructure change during development of pistils of litchi male and female flower. The results showed that at the megasporocyte stage of female flowers, calcium precipitates was located mainly at cell wall and intercellular space of inner integument near the micropyle and style cells, and to a lesser extent in vacuoles. Vascular tissues also contained much calcium precipitates. In inner integument cells near the micropyle of male flowers, the vacuole contained most of the calcium precipitates. Calcium precipitates in style cell and vascular tissues of male flowers was sparse and seldom seen. After meiosis of megasporocyte, pistils of female flowers continued to grow and those of male flowers aborted. In female flowers, calcium precipitates concentration became lower and calcium precipitates was probably transported to the places for future pollen bourgeoning and fertilization. Cell wall calcium precipitates concentration increased in the inner integument cells near the micropyle. Calcium precipitates concentration increased from topper style cells to lower ones. In male flowers, inner integument cells near the micropyle underwent the programmed cell death (PCD): flow of calcium from vacuoles into nucleus might had triggered the PCD process. A continuous channel was formed between perinuclear space and cytoplasm membrane lumen, and calcium flowed freely between nuclear membrane and plasma membrane. At certain time and locations, calcium precipitates was newly appeared at some organelles like endoplasimic reticulum, mitochondria and peroxisomes. This calcium redistribution in cells might trigger and regulate the process of PCD. In male flowers, style cells containing no calcium precipitation soon began to degenerate.

Identification of polysaccharides from pericarp tissues of litchi (Litchi chinensis Sonn.) fruit in relation to their antioxidant activities.

A large number of polysaccharides are present in the pericarp tissues of harvested litchi fruits. A DEAE Sepharose fast-flow anion-exchange column and a Sephadex G-50 gel-permeation column were used to isolate and purify the major polysaccharides from litchi fruit pericarp tissues. Antioxidant activities of these major polysaccharide components were also evaluated. An aqueous extract of the polysaccharides from litchi fruit pericarp tissues was chromatographed on a DEAE anion-exchange column to yield two fractions. The largest amount of the polysaccharide fraction was subjected to further purification by gel filtration on Sephadex G-50. The purified product was a neutral polysaccharide, with a molecular weight of 14 kDa, comprised mainly of 65.6% mannose, 33.0% galactose and 1.4% arabinose. Analysis by Smith degradation indicated that there were 8.7% of (1-->2)-glycosidic linkages, 83.3% of (1-->3)-glycosidic linkages and 8.0% of (1-->6)-glycosidic linkages in the polysaccharide. Furthermore, different polysaccharide fractions extracted and purified from litchi fruit pericarp tissues exhibited strong antioxidant activities. Among these fractions, the purified polysaccharide had the highest antioxidant activity and should be explored as a novel potential antioxidant.

[Changes in cell ultrastructure during sexual determination of litchi staminate flower].

The ultrastructural changes of meristematic cell during the degeneration of gynoecium primordium leading to the formation of staminate flower of litchi were followed. Degradation of the cells and transport of the dissolved cytoplasmic components were well ordered. Configurations of rough endoplasmic reticulum (RER) changed significantly. ER played an important role in degenerative processes of gynoecium primordiuml cells. The degenerative processes started with the appearance of long RER cisternae throughout the cytoplasm. Some long RER cut or enclosed the cytoplasm. Some RER connected nucleus and mitochondria of adjacent cells, formed a ridge-like connection. Later the RER formed concentric patterns and then became irregular stacks. RER and golgiosome produced many vesicles, which were importance to protoplasmic degradation and intercellular transport of the cellular debris. The number of mitochondria increased up to the time when they began to degrade in batches. Peroxisomes appeared temporarily at the middle stage near the nucleus. The nucleolus disintegrated at the beginning of degeneration of nucleus. Then fragments of chromatin aggregated at the periphery of nuclear membrane and diffused outward. In some nuclei the perinuclear membrane became dilated and puffs were formed. As cell degeneration progressed, the protoplasm disintegrated and dissipated in an orderly fashion, i.e. ribosomes became disorganized first, followed by peroxisomes, ER, golgiosoms, mitochondria and nucleus. Eventually, gynoecium primordium cells digested all of the cytoplasm, leaving only cell wall with high electron density. Most of the products of degeneration of gynoecium primordium cells were removed through either symplastic or apoplastic pathways. Programmed cell death (PCD) may be involved in the degeneration of meristematic cells at the gynoecium primodium.