Exploring the dynamism of
chloroplast & mitochondrial nucleoids
西村芳樹 Dr. Yoshiki Nishimura
yoshiki(at)pmg.bot.kyoto-u.ac.jp
Dynamism of chloroplast /mitochondrial nucleoids
"Chromosomes" within chloroplasts & mitochondria
According to the intracellular symbiosis theory, chloroplasts and mitochondria are presumed have derived from independent bacteria, closely related to cyanobacteria and alpha-proteobacteria, respectively. Reflecting their evolutionary history, chloroplasts and mitochondria have their own "chloroplast/mitochondrial genome". The chloroplast/mitochondrial genome encodes many photosynthetic genes and genes involved in respiration, and as the center of photosynthesis and respiration, it supports life on Earth. The chloroplast/mitochondrial genome binds to a variety of proteins and folds to form a structure called the "chloroplast/mitochondrial nucleoid". The chloroplast/mitochondrial nucleoids plays a central role in DNA replication, repair, gene expression, and inheritance, but its detailed structure and regulatory mechanisms are still shrouded in mystery.
Chloroplast nucleoids in motion - Moments of Chloroplast nucleoid division captured by the microfluidic device
The chloroplast nucleoids behave dynamically. We have successfully labeled chloroplast nucleoids with fluorescent proteins and followed their behavior in living cells by observing them with the microfluidic devices.
In the green alga Chlamydomonas that we are focusing on, chloroplasts are usually observed as 5-10 spherical structures per chloroplast. In this study, we observed, for the first time, the dynamic motion of these spherical chloroplast nucleoids, which are disassembled and transformed into interconnected network-like structures, prior to the chloroplast division and reverted to spherical structures after the completion of chloroplast division.
Figure １ Division of chloroplast nucleoids captured with the microfluidic device.
(Kamimura et al., Comms. Biol. 2018; Yomiuri news（2018.5))
Scissors to cut the chloroplast nucleoids ~ Discovery of the chloroplast-type Holliday junction resolvase ~
Chloroplast DNA (chloroplast DNA) must be faithfully inherited in order for chloroplasts to divide and proliferate. In the unicellular green alga Chlamydomonas, chloroplast nucleoids can be visualized in living cells using YFP (Fig. 2: yellow bright spots superimposed on chloroplast red autofluorescence). In wild type cells, the chloroplast nucleoids are dispersed and evenly distributed among the four divided chloroplasts (top left: arrowhead). However, in the monokaryotic chloroplast (moc)1 mutant, only one chloroplast monopolizes most of the chloroplast nucloeids (top right: arrow head). We found that moc1 mutant lacked the enzyme, Holliday junction resolvase, that separate chloroplast DNA precisely, like 'scissors'. The lower panel of figure 2 shows how this enzyme binds to the center of an artificial Holliday junction (lower left: cross structure) made of DNA oligami (center: arrow) and cleaves it (right: arrow), as visualized by a high-speed atomic force microscope. Many plants on Earth possess this enzyme, which controls the distribution of chloroplast nucleoids and supports photosynthesis and chloroplast biosynthesis.
Fig. 2 Unequal distribution of chloroplast nucloeids (yellow bright spots: arrows) in Holliday junction resolvase deficient mutants (moc: top panel); AFM images showing the binding of chloroplast Holliday junction resolvase to the center of Holliday Junction (center) and symmetrical cleavage (right) as visualized by DNA oligami and fast atomic force microscopy techniques. (Kobayashi et al., Science 2017)
Mystery of maternal inheritance
Both males and females have chloroplasts/mitochondria. But in many cases, the male ones do not pass on to the offspring, but only the female ones to the offspring. This is called maternal inheritance, and the mechanism by which it occurs has been debated for more than 100 years since its first report in 1909. We would like to answer this question through genetic, cytological, and molecular biological analysis of Chalmydomonas, a single-celled green alga.
In Chlamydomonas, a single-celled green alga, male and female gametes have exactly the same shape, each contributing the same amount of mitochondrial (chloroplast) DNA to the offspring. Nevertheless, chloroplast DNA is inherited maternally and mitochondrial DNA is inherited paternally. Staining of live zygotes with the DNA-specific fluorescent dye SYBR Green I revealed that the male chloroplast nucleoids were degraded only 45-60 minutes after mating (Fig. 3). Furthermore, the degradation of uniparental mitochondrial (mt) nucleoids has been shown to occur in the sperm of Medaka fish (Nishimura et al., PNAS 2006), slime mold, and the basidiomycete Cryptococcus, suggesting that the active degradation of uniparental mitochondrial & chloroplast nucleoids would be one of the fundamental mechanisms of maternal inheritance.
We are now working on the isolation of mutants and reverse genetic analysis of zygote-specific genes in order to identify the genes responsible for the degradation of male chloroplast and mt nucleoids. We found that the active degradation of uniparental chloroplast nucleoids is regulated by the gametogenesis-specific transcription factors GSP1-GSM1 (Nishimura et al., Plant Cell 2012). In Cryptococcus, we found that the mt nucleoids of one parent are selectively degraded, followed by the elimination of mitochondria lacking mt nucleoids by autophagy (Nishimura et al., Sci Rep. 2020). In the future, we would like to elucidate the genes that regulate maternal inheritance in order to gain a better understanding of the overall mechanism of maternal inheritance.
Fig. 3 Selective degradation of the male chloroplast nucleoids. Of the chloroplast nucleoids (arrows) observed in male and female chloroplasts immediately after mating (A), those of male origin (right) completely disappeared within 60 min after zygote formation (B).
*If you are interested, please contact yoshiki(at)pmg.bot.kyoto-u.ac.jp