BOP genes and sac-like structures of Callicarpa saccata

Callicarpa saccata is a small tree originating from Borneo in Southeast Asia. Its name comes from the sac-like structure at the leaf base. This structure is unique in the Callicarpa genus. Hence its developmental background is of particular interest. Previous findings in a model plant, Arabidopsis thaliana and C. saccata suggest that BLADE-ON-PETIOLE (BOP) genes could be regulating sac formation. The study described in this article aimed to support or falsify the causality of a nonsense BOP mutation to sac formation by comparing the BOP gene sequences between C. saccata and other Callicarpa species. Results show that there are at least two copies of BOP genes in C. saccata. The copy identified in this study does not contain a nonsense mutation and is highly conserved within the genus. Introduction Callicarpa is a genus of shrubs and small trees distributed in east and Southeast Asia, Australia, southeast North America and Central America. Its name refers to “beauty berry” in Latin, as fruits of Callicarpa show distinctive pink to red-purple colors with a metallic luster. Borneo is the largest island of Asia and has one of the oldest rainforests in the world. The Borneo rainforest is home to many species of plants and animals, which makes it an important resource for biological researches. Up to 2008, 23 Callicarpa species had been identified in Borneo, among which Callicarpa saccata is of particular interest for its distinctive sac-like structures at the base of leaves. No other species of the Callicarpa genus has been observed to have a similar structure. In the wild, the sac-like structures are observed to be domatia occupied by ants (Figure 1a). Secretions inside the sacs have been discovered to be potentially nutritious for ants (Ezaki, Saawada, Hirai and Tsukaya, unpublished). Therefore it has been hypothesized that C. saccata and ants work in mutualism: C. saccata provides shelter and food for ants, while ants defend the plant from herbivores and parasites. Moreover, the sac-like structures develop normally even when C. saccata is grown free of ants in the lab (Figure 1b), which means that the structure is genetically coded and is not caused by ant occupation. a b Figure 1. The morphology of sac-like structures (a) in the wild and (b) in the lab. (a) was taken by Professor Hirokazu Tsukaya in Borneo and (b) was taken by Ms. Kazune Ezaki in the Botanical Gardens, the Univ. Tokyo. BLADE-ON-PETIOLE (BOP) genes have been hypothesized to be responsible for the unusual development of the sac-like structures. BOP loss-of-function mutants in Arabidopsis exhibit excessive growth around petiole regions, which is reminiscent of sac-like structures on C. saccata. Previous studies showed C. saccata was different from C. involucrata by having a single nonsense mutation instead of a serine in one of the BOP genes of C. saccata (Figure 2). The mutation takes place in the ANK domain, which is known for protein-protein interactions. Therefore the resulted truncated BOP protein could be dysfunctional and causing overgrowth at the leaf base. Effort was also made to sequence another copy of BOP gene in C. saccata. However, only a small range of BOP2 was sequenced which did not include the location corresponding to the nonsense mutation in the first BOP gene. Hence we are interested in sequencing more copies of BOP genes and compare the sequences of C. saccata with that of other Callicarpa species to analyze the causal relationship of the nonsense mutation to the sac-like structure. Figure 2. A nonsense mutation was discovered in a copy of BOP gene of C. saccata, 6 which is marked by the black circle. Methods Five Callicarpa species were involved: C. saccata, C. involucrata, C. glabrifolia, C. pentandra and C. × shirasawana. The first four species all originate from Borneo 3 while the last one is a natural hybrid between two Callicarpa species and is native to Japan . Genomic DNA was isolated using DNeasy Plant Mini Kit (Qiagen). Three pairs of degenerate primers, ranging from 33 to 36 nucleotides, had been designed by previous researchers. Nested Touchdown PCR was performed by amplifying first with primers F1 and R1 and second with F1 and R2. The polymerase kit used was KOD Plus (Toyobo). The PCR product mixture was separated by electrophoresis and candidate bands were extracted using QIAquick Gel Extraction Kit (Qiagen). The purified amplicons were sequenced with the same primers by the company (Fasmac Sequence Service). The sequencing results were analyzed using CLC Main Workbench and were compared with Arabidopsis BOP genes from the NCBI gene database. Cloning after one round of PCR amplification was also tried out but did not give sequencing results covering the region of interest. Results and discussion BOP sequences of all species except for C. pentandra were successfully sequenced and did not show a nonsense mutation at the same serine (Figure 3). No other nonsense mutation was observed throughout the sequences either. The sequencing results were considered reliable because the sequence from each species could be verified twice with forward and reverse primers. In addition, the chromatograms were clear around the region of interest (not shown). Therefore it is further confirmed that C. saccata has at least two copies of BOP genes. The copy of BOP gene sequenced here is highly conserved across Callicarpa species. Figure 3. There was no amino acid mutation at the serine of interest, which is also uniform across all four species. The left column marked by the black box represents the serine that is mutated in the previous study. Apart from the main finding, it was also discovered that the BOP gene of C. × shirasawana has two single amino acid substitutions compared with the three Bornean species (not shown). This difference agrees with the fact that C. × shirasawana has a very different geographic distribution with Bornean species. There is no final conclusion to the causality of nonsense mutation to the sac development. Future experiments need to be performed to sequence the copy of BOP gene corresponding to the nonsense mutation in other Callicarpa species. The hypothesis that the nonsense mutation causes sac-like structures could be supported if other Callicarpa species do not have the same mutation. It is also possible to try to establish the transgenic system in C. saccata, so that full BOP genes can be overexpressed in C. saccata or BOP genes could be knocked down in other Callicarpa species to observe disappearance or development of sac-like structures. Acknowledgement I would like to express my gratitude to the University of Tokyo Research Internship Program for offering such a great summer research opportunity. I am also grateful that Professor Tsukaya accepted me to his lab and guided me into the world of plants. All lab members have been greatly kind and helpful. Especially I would like to thank Ms. Kazune Ezaki, Ms. Pin Guo, Dr. Keiko Sakakibara, Mr. Koro Hattori and Mr. Xiaofeng Yin for offering advice when I faced difficulties in the experiments.