High-level expression of a novel chromoplast phosphate transporter ClPHT4;2 is required for flesh color development in watermelon.

Chromoplast development plays a crucial role in controlling carotenoid content in watermelon flesh. Modern cultivated watermelons with colorful flesh are believed to originate from pale-colored and no-sweet progenitors. But the molecular basis of flesh color formation and regulation is poorly understood. More chromoplasts and released carotenoid globules were observed in the red-fleshed fruit of the 97103 cultivar than in the pale-colored fruits of the PI296341-FR line. Transcriptome profiles of these two materials identified Cla017962, predicted as ClPHT4;2, was dramatically up-regulated during flesh color formation. High ClPHT4;2 expression levels were closely correlated with increased flesh carotenoid contents among 198 representative watermelon accessions. Down-regulation of ClPHT4;2 expression in transgenic watermelons reduced the fruit carotenoid accumulation. ClPHT4;2 as a function of chromoplast-localized phosophate transporter was tested by heterologous expression into a yeast phosphate-uptake-defective mutant, western blotting, subcellular localization, and immunogold electron microscopy analysis. Two transcription factors, ClbZIP1 and ClbZIP2, were identified, which responded to ABA and sugar signaling to regulate ClPHT4;2 transcription only in cultivated watermelon species. Our findings suggest that elevated ClPHT4;2 gene expression is necessary for carotenoid accumulation, and may help to characterize the co-development of flesh color and sweetness during watermelon development and domestication.

Ultramicroscopy reveals that senescence induces in-situ and vacuolar degradation of plastoglobules in aging watermelon leaves.

The dynamics of plastoglobules in chloroplasts in aging watermelon leaves were examined by means of transmission electron microscopy, with the aim to understand the intracellular sites for the degradation of plastoglobules in response to leaf senescence. Plastoglobules in chloroplasts in aging leaves with 40% loss of chlorophyll increased drastically in number and size in comparison with young and mature leaves. As senescence advanced, plastoglobules underwent degradation within chloroplasts, or were secreted outside chloroplasts. There were two distinct types of secretion. One type was that chloroplasts protruded to form plastoglobule-containing vesicles and, as the vesicles were detached from chloroplasts, plastoglobules were carried outside chloroplasts. The other type was that plastoglobules squeezed out through the chloroplast envelope into cytoplasm. Lipid droplets were present in the vacuole and underwent degradation therein. Lipid droplets in the vacuole shared similar ultramicroscopic appearance with plastoglobules in chloroplasts, indicating that plastoglobules were engulfed and degraded by the vacuole after they were secreted outside chloroplasts. These results suggested that senescence induces both in-situ and vacuolar degradation of plastoglobules in aging watermelon leaves.

Ultrastructural study on dynamics of plastids and mitochondria during microgametogenesis in watermelon.

Dynamics of plastids and mitochondria during microgametogenesis in watermelon were examined by means of transmission electron microscopy. Plastids are present as proplastids in the microspore and as amyloplasts in the vegetative cell of the bicellular pollen grain, whereas the generative cell is completely devoid of plastids, suggesting that microspore plastids are excluded from the generative cell during the microspore mitotic division. Therefore, watermelon is classified as Lycopersicon type, where plastids exclusion from the generative cell leads to purely maternal plastid inheritance. Mitochondria in the generative cell show noticeable alterations in size and cristae during microgametogenesis. The diameter of mitochondria is about 0.5 µm in the newly born generative cell, while only about 0.16 µm in the spindle-shaped generative cell. Numerous cristae are present in mitochondria in the spherical generative cell, but, in contrast, mere two or three cristae retain in the spindle-shaped generative cell in the mature pollen grain. In conclusion, the size and cristae number of mitochondria in the generative cell are reduced significantly during microgametogenesis in watermelon.

Effects of fruit maturity on watermelon ultrastructure and intracellular lycopene distribution.

This study employed transmission electron microscopy (TEM) to conduct research on the ultrastructure of watermelon (cultivar: Hazera SW1) mesocarp samples of different maturities. Micrographs from immature fruit showed incompletely formed chromoplasts. A combination of distinct pigment-bearing chromoplasts and incompletely formed chromoplasts was observed in mature watermelon micrographs. Electron micrographs showed chromoplasts changing from a less organized globular form in immature to a symmetrical form in mature to an asymmetrical form in overmature watermelons. This study furthers our understanding of watermelon physiology and the effect of maturity on compartmentalization of lycopene.

Interaction of sodium dodecyl sulfate with watermelon chromoplasts and examination of the organization of lycopene within the chromoplasts.

The properties of plant-derived precipitates of watermelon lycopene were examined in aqueous sodium dodecyl sulfate (SDS) as part of an ongoing effort to develop simpler, more economical ways to quantify carotenoids in melon fruit. Levels of SDS >0.2% were found to increase the water solubility of lycopene in the state in which it was isolated from watermelon. Electron microscopy and chemical analyses suggested that the watermelon lycopene as isolated is packaged inside a membrane to form a chromoplast. Spectral peaks in the visible region of the watermelon chromoplasts in SDS exhibited a bathochromic shift from those in organic solvent. Watermelon chromoplasts in SDS exhibited pronounced circular dichroic activity in the visible region. Binding measurements indicated that about 120 molecules of SDS were bound per molecule of lycopene inside the chromoplast; likely, the detergent molecules are bound to the chromoplast membrane. Around 80% of the chromoplast-SDS complexes were retained on a 0.45 mum membrane filter. Together, these observations are consistent with lycopene in a J-type chiral arrangement inside a membrane to form a chromoplast. The binding of SDS molecules to the chromoplast membrane form a complex that is extensively more water-soluble than the chromoplast alone.

[The ultrastructural study of synergids before and after fertilization in watermelon embryo sacs].

The ultrastructure of synergids of watermelon (Citrullus Lanatus L.) was investigated using transmission electron microscopy at following stages of embryo sacs: 1. Unpollination, on the first flowering day. 2. Unpollination, on 2nd day after anthesis (DAA). 3. Fertilization, on DAA 2. The synergids with distinct filiform apparatus at the micropylar end have abundant organelle, such as mitochondria, endoplasmic reticulum, and plastids in cytoplasm, which indicate that they are active on the first flowering day. No wall is present at the chalazal part of synergid, and there are some flocculent materials and vesicles in the spaces of cytoplasma membranes among synergid, egg cell and central cell in embryo sacs at the first and the second stages. On DAA 2, in unpollinated embryo sacs, the central large vacuole of synergid is divided into several smaller ones and the starch grains decrease in cytoplasm. There is no newly synthesized wall at the chalazal end of persistent synergid in fertilized embryo sacs. The contents of degenerated synergid, in the form of electron dense granules, are located in the wide space among central cell, zygote and persistent synergid, and some of them migrate into central cell through cytoplasma membrane. Therefore, it is deduced that the contents of synergid might serve as a nutrient supplement to the development of endosperm, but not embryo.