Differential Expression of Carotenoid Biosynthesis Genes among Different Colored Flesh in Watermelons [Citrullus lanatus (Thunb)]

Carotenoids are plant compounds that serve a variety of essential functions in the plant and have also been found to have several health-promoting activities in humans. Carotenoids found in watermelon (Citrullus lanatus) flesh are responsible for the various colors such as red, yellow and orange. Previous inheritance studies of flesh color revealed that six genes were involved in color determination. The relationship and interaction of these genes suggests that some color-determining genes may be the result of mutations on the structural genes encoding enzymes in the carotenoid biosynthesis pathway. In this study we were able to isolate and sequence six genes encoding enzymes involved in the carotenoid biosynthetic pathway, and determine their expression in different colored watermelon fruit. The cDNA was synthesized from total RNA using RACE (Rapid Amplification of cDNA ends) kit (SMART RACE cDNA Amplification Kit; Clontech, Palo Alto, Calif.). Degenerate primers were designed based on published homologous genes from other species and were used to isolate gene fragments and full-length cDNAs of phytoene synthase, phytoene desaturase, _-carotene desaturase, β-cyclase, β-carotene hydroxylase and zeaxanthin expoxidase. RT-PCR was carried out to examine any differential expression of cloned genes in white, yellow, orange and red-fleshed watermelon. All cloned enzyme-encoding genes were expressed regardless of flesh colors. These results indicate that carotenoid biosynthesis may be regulated at the post-transcriptional level. One interesting feature supports this hypothesis. In case of β-cyclase, a 229-bp leader intron was identified, and an unspliced mRNA with this leader intron existed dominantly in cDNA pool of all samples.

Cis-regulation downstream of cell type specification: a single compact element controls the complex expression of the CyIIa gene in sea urchin embryos

CyIIa, a cytoskeletal actin gene of Strongylocentrotus purpuratus, is expressed specifically though transiently in the embryonic skeletogenic and secondary mesenchyme and, later in development, is permanently activated in the hindgut and midgut. CyIIa transcription follows, and is therefore downstream of, the initial specification of these embryonic domains. A detailed functional analysis of the cis-regulatory system governing the rate and the location of CyIIa expression during development was carried out using GFP expression constructs. About 4.4 kb of CyIIa sequence including a leader intron were examined for cis-regulatory function. Distal elements scattered over several kb account for 60% of the quantitative output of the expression construct and a strong amplifier of expression is located within the leader intron. However, the complex spatial pattern of CyIIa expression is completely reproduced by a compact upstream regulatory element <450 bp in length. We found no evidence anywhere in the 4.4 kb sequence examined for negative regulators required to repress ectopic expression. The specific site that mediates CyIIa expression in the midgut in late embryos and larvae was identified. This site is the same as that necessary and sufficient for midgut expression of the Endo16 gene late in development, and was shown to bind the same transcription factor. Except for some temporal and quantitative features, the S. purpuratus expression construct is expressed accurately and specifically in the same diverse cell types when introduced into embryos of Lytechinus pictus, which belongs to a different echinoid order. No ectopic expression was observed, in contrast to the result of a similar interspecific gene transfer experiment carried out earlier on a different cytoskeletal actin gene that is expressed much earlier in development. Presentation of the set of transcription factors that activate CyIIa in the differentiated cells in which it is expressed is apparently a conserved feature of these cell types.