A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom    

Abstract

Research in yeast and animals has resulted in a well-supported consensus model for eukaryotic cell cycle control. The fit of this model to early diverging eukaryotes, such as the plant kingdom, remains unclear. Using the green alga Chlamydomonas reinhardtii, we developed an efficient pipeline, incorporating robotics, semiautomated image analysis, and deep sequencing, to molecularly identify >50 genes, mostly conserved in higher plants, specifically required for cell division but not cell growth. Mutated genes include the cyclin-dependent kinases CDKA (resembling yeast and animal Cdk1) and the plant-specific CDKB. The Chlamydomonas cell cycle consists of a long G1 during which cells can grow >10-fold, followed by multiple rapid cycles of DNA replication and segregation. CDKA and CDKB execute nonoverlapping functions: CDKA promotes transition between G1 and entry into the division cycle, while CDKB is essential specifically for spindle formation and nuclear division, but not for DNA replication, once CDKA-dependent initiation has occurred. The anaphase-promoting complex is required for similar steps in the Chlamydomonas cell cycle as in Opisthokonts; however, the spindle assembly checkpoint, which targets the APC in Opisthokonts, appears severely attenuated in Chlamydomonas, based on analysis of mutants affecting microtubule function. This approach allows unbiased integration of the consensus cell cycle control model with innovations specific to the plant lineage.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in <em>Chlamydomonas reinhardtii</em>

“…This regulatory scheme provides a natural distinction between genes primarily required for cell growth and integrity and genes required specifically for the cell division cycle. We found that the 10-hr and 20-hr time points are very informative for an initial rough phenotypic cut 1 . The broad classes of ts lethal mutants that we recognize currently, based on these images (see S.I.…”

“…The "Notch" and "Popcorn" phenotypes were shown previously to be characteristic of most cell cycle-specific lesions (e.g., mitotic cyclin-dependent kinase, DNA replication machinery, and Topoisomerase II) 1 . The appearance of one (Notch) or multiple (Popcorn) apparent planes of incipient but unsuccessful cell division is a convenient morphological indicator of cell cycle initiation.…”

“…The broad classes of ts lethal mutants that we recognize currently, based on these images (see S.I. in Tulin and Cross, 2014) 1 , are: Notch, Popcorn, Round, Small, Medium, early lysis, and multiple-cycle (Figure 8).…”

“…This problem can be dealt with to some extent by comparing multiple alleles; if they all share a common phenotype, this is more likely to reflect the result of simple inactivation of the gene. Multiple alleles are also very helpful for definitive molecular identification of the causative lesion 1 . However, the "frequent flyer" problem means that multiple alleles in rarely hit genes can be difficult to recover.…”

“…Phenotypic analysis at high temperature then provides immediate information about the function of the mutated gene. We reported on the isolation and characterization of ts lethal mutations in ~70 essential genes in Chlamydomonas, focusing especially on genes involved in cell cycle progression and control 1,4 . Ts lethal screens have been a mainstay of genetic analysis in microorganisms for decades 5,6 .…”