Pigmentation and TYRP1 expression are mediated by zinc through the early secretory pathway-resident ZNT proteins.

Tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TYRP1 and TYRP2) are essential for pigmentation. They are generally classified as type-3 copper proteins, with binuclear copper active sites. Although there is experimental evidence for a copper cofactor in TYR, delivered via the copper transporter, ATP7A, the presence of copper in TYRP1 and TYRP2 has not been demonstrated. Here, we report that the expression and function of TYRP1 requires zinc, mediated by ZNT5-ZNT6 heterodimers (ZNT5-6) or ZNT7-ZNT7 homodimers (ZNT7). Loss of ZNT5-6 and ZNT7 function results in hypopigmentation in medaka fish and human melanoma cells, and is accompanied by immature melanosomes and reduced melanin content, as observed in TYRP1 dysfunction. The requirement of ZNT5-6 and ZNT7 for TYRP1 expression is conserved in human, mouse, and chicken orthologs. Our results provide novel insights into the pigmentation process and address questions regarding metalation in tyrosinase protein family.

Zinc transport via ZNT5-6 and ZNT7 is critical for cell surface glycosylphosphatidylinositol-anchored protein expression.

Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs (ZNT5-ZNT6 heterodimers [ZNT5-6] and ZNT7-ZNT7 homodimers [ZNT7]), which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway.

Phylogeography of the genus Cardiandra based on genetic variation in cpDNA sequences.

We investigated the phylogenetic relationships within the genus Cardiandra based on plastid DNA sequences. The phylogenetic tree showed that Cardiandra populations from the Ryukyu Islands (Japan) and Taiwan were monophyletic (Ryukyu-Taiwan clade), whereas taxa from China and mainland Japan were sisters to this clade. The divergence time between the Ryukyu-Taiwan clade and the other species was estimated to be 0.082 MYA, i.e., the late Pleistocene. The infrageneric and/or infraspecific differentiation of Cardiandra is estimated to have depended largely on allopatric differentiation caused by the presence or division of the past landbridge of the Ryukyu Islands, which connected mainland Japan to the Asian Continent during the Quaternary.

Mode of pollen-tube growth in Pistils of Myrica rubra (Myricaceae): a comparison with related families.

BACKGROUND AND AIMS: It is generally known that fertilization is delayed for more than a few weeks after pollination in Fagales. Recent studies showed that, during that period, pollen tubes grew in pistils in close association with the development of the ovule in a five-step process in Casuarina (Casuarinaceae) and a four-step process in Alnus (Betulaceae). The number of pollen tubes was reduced from many to one, a fact suggesting that delayed fertilization plays a role for gametophyte selection. Myrica (Myricaceae) also shows delayed fertilization for >2 weeks after pollination, but nothing is known of how pollen tubes grow in the pistil during that period. METHODS: Pollen-tube growth and the development of the ovule in pistils was investigated by fluorescent and scanning electron microscopy and analysis of microtome sections of the pistils. KEY RESULTS: Developmental study of the pollen-tube growth in the pistil of M. rubra showed that the tip of the pollen tube was branched or lay in a zigzag pattern in the upper space of the ovarian locule or near the tip of the integument, and subsequently was swollen on the nucellar surface. Such morphological changes indicate that the pollen-tube growth was temporarily arrested before fertilization. The pollen-tube growth in M. rubra can therefore be summarized as occurring in three steps: (1) from the stigma to the ovarian locule; (2) from the ovarian locule to the nucellar surface; and (3) from the nucellar surface to the embryo sac. CONCLUSION: Myrica differs from other families in that the pollen tubes arrest their growth on the nucellar surface, probably digesting nutrient from nucellar cells. There is little information on five other families of Fagales. An extensive study is needed to better understand the diversity and function of the mode of pollen-tube growth within the order.

Delayed fertilization and pollen-tube growth in pistils of Fagus japonica (Fagaceae).

In contrast to most angiosperms, in which fertilization occurs 1 or 2 days after pollination, in some plant orders, including the Fagales, fertilization is delayed from 4 days to more than 1 year, raising questions regarding why fertilization is delayed and where and how pollen tubes remain in the pistil during the delay. To answer these questions, we investigated pollen-tube growth in pistils of Fagus japonica (Fagaceae), which are tricarpellate and have six ovules, using light, fluorescence, and scanning electron microscopy. The ovules were immature at the time of pollination and required 5 weeks to become fully developed. During this 5 weeks, pollen tubes grew from the stigma to the embryo sac in association with the development of ovules and intermittently in three steps with two growth-cessation sites, i.e., on the funicle and near the micropyle. The number of pollen tubes was gradually reduced from many to one at the two growth-cessation sites, and fertilization occurred in one ovule that apparently developed earlier than the others in the pistil. Thus, delayed fertilization plays an important role in gametophyte competition and selection leading to nonrandom fertilization. Intermittent pollen-tube growth is also likely widespread in angiosperms because it is known in other Fagales and an unrelated order Garryales.

Intermittent pollen-tube growth in pistils of alders (Alnus).

In alders, where fertilization occurs approximately 8 weeks after pollination, the pollen tube (male gametophyte) grows intermittently in four steps in close association with the development of the ovary and its ovules. Pollen tubes stop growing in the style, at the ovarian locule, and at the chalaza (ovule), before reaching an embryo sac for fertilization. At the stage when the ovary develops an ovule primordium in each of the two locules, many pollen tubes germinate on the stigma, and a few of them reach the style, where they remain for approximately 7 weeks. Thereafter, a single tube resumes growing; with a short stop in the upper space of the ovarian locule, it reaches the older of the two ovules when it has developed a two-nucleate embryo sac. Except in the last step, where the tube grows from the chalaza to an embryo sac (female gametophyte), an eight-nucleate mature embryo sac is not necessary for pollen-tube guidance in the pistil. Although the intermittent pollen-tube growth appears to play an important role in the selection of a single pollen tube from many and one ovule from two, its detection provides insight into the study of the mechanism of pollen-tube guidance.

Pollen-tube growth and fertilization mode in Gymnostoma (Casuarinaceae): their characteristics and evolution.

A unique mode of fertilization called "chalazogamy", whereby the pollen tube passes through the chalaza instead of the micropyle, is known in several species of derived genera in Casuarinaceae. In this paper we report the occurrence of chalazogamy in Gymnostoma ( G. poissonianum), the most primitive genus in the family. We also show that the pollen tube grows discontinuously from the stigma to ovules in about 3 months. At the time of pollination, the ovules have not yet formed in the ovary, and require a long time to develop. The pollen tube(s) lie in a zigzag line and are branched in the upper region of the style, and their growth is arrested there until the ovary develops further. Studies of the relevant literature further revealed discontinuous pollen-tube growth in relation to a prolonged period between pollination and fertilization, as well as chalazogamy, in Betulaceae, Juglandaceae and/or Fagaceae that are closely related to Casuarinaceae. This feature may have derived early in the evolution of Fagales.

Pollen-tube growth pattern and chalazogamy in Casuarina equisetifolia (Casuarinaceae).

For a better understanding of pollen-tube guidance in relation to pollen-pistil interaction, we investigated the mode of pollen-tube growth in pistils of Casuarina equisetifolia, a monoecious, wind-pollinated species that undergoes chalazogamous fertilization. The pistil is bicarpellate, but only one of the two carpels develops with two ovules. One of these ovules develops more than four embryo sacs. Pistils usually require more than 1 month to reach maturity after pollen grains have been deposited on the stigmas. During that period, pollen-tube growth proceeds discontinuously in five distinct steps that lead up to fertilization: (1) from the stigma to the upper region of the style, (2) from the upper region of the style to a septum in the ovary, (3) from the septum to the surface of the funiculus, (4) from the funiculus to chalaza in the ovule, and (5) from the chalaza to an egg apparatus. Probably because of competitive interaction between male and female gametophytes (or ovules), one pollen tube is selected from among many during the first step (just before the second step), one ovule from the two during the second and third steps, and one embryo sac from more than four during the fourth and fifth steps. On the basis of our results, erroneous drawings and explanations reported in earlier publications on chalazogamy in Casuarinaceae should be brought into question.