Unequal contribution of two paralogous CENH3 variants in cowpea centromere function

Abstract

In most diploids the centromere-specific histone H3 (CENH3), the assembly site of active centromeres, is encoded by a single copy gene. Persistance of two CENH3 paralogs in diploids species raises the possibility of subfunctionalization. Here we analysed both CENH3 genes of the diploid dryland crop cowpea. Phylogenetic analysis suggests that gene duplication of CENH3 occurred independently during the speciation of Vigna unguiculata. Both functional CENH3 variants are transcribed, and the corresponding proteins are intermingled in subdomains of different types of centromere sequences in a tissue-specific manner together with the kinetochore protein CENPC. CENH3.2 is removed from the generative cell of mature pollen, while CENH3.1 persists. CRISPR/Cas9-based inactivation of CENH3.1 resulted in delayed vegetative growth and sterility, indicating that this variant is needed for plant development and reproduction. By contrast, CENH3.2 knockout individuals did not show obvious defects during vegetative and reproductive development. Hence, CENH3.2 of cowpea is likely at an early stage of pseudogenization and less likely undergoing subfunctionalization.

Epigenetically mismatched parental centromeres trigger genome elimination in hybrids

“…Could GFP-ts be removed selectively while CENH3 persisted? Previous reports are consistent with the possibility of wild-type CENH3 persistence ( 12 , 25 , 40 ). To compare the stability of GFP-ts and wild-type CENH3, we examined the immature (flower stages −2 and −1) and mature (flower stages +1 and +2) ( 41 ) ovules bracketing normal self-fertilization time ( Fig.…”

Only the Rye Derived Part of the 1BL/1RS Hybrid Centromere Incorporates CENH3 of Wheat

“…The precise assembly of the kinetochore complex at the centromere is epigenetically determined by substituting a centromere-specific histone H3 variant CENH3 for histone H3 in centromeric nucleosomes ( Henikoff and Dalal, 2005 ; Allshire and Karpen, 2008 ). Besides some diploid species encoding multiple functional CENH3 variants, e.g., barley ( Sanei et al, 2011 ; Ishii et al, 2015 ), rye ( Evtushenko et al, 2017 , 2021 ), Fabeae species ( Neumann et al, 2015 ), and cowpea ( Ishii et al, 2020 ), most diploid eukaryotes encode only one variant of CENH3. In contrast, in allopolyploids species like wheat ( Yuan et al, 2015 ), each parental subgenome possesses at least one CENH3 variant operating in the context of multiple species-specific centromeric sequences.…”

Expression of Two Rye CENH3 Variants and Their Loading into Centromeres

“…Not aware of any similar quantitative assessments in plants, we propose that the four patterns of polynucleosomal CENH3 subdomains form part of centromeres. A similar subdomain organization of centromeric chromatin with alternating nucleosomal clusters containing both CENH3 variants and nucleosomes containing the canonical histone H3 was described for centromeres in barley [16], legumes [38,41], humans and Drosophila [42,43]. Thus, the organization of chromatin with polynucleosomal subdomains containing a unique set of histone variants is evolutionarily conserved for centromeres and makes centromeric chromatin different from the neighboring noncentromeric regions [41][42][43].…”

“…It is possible that new approaches to studying and assessing functional divergence will help reveal the above functions in cereals-in addition to conservation. The study of centromeric functions of the CENH3 variants in cowpea through the CRISPR/Cas9-based inactivation of one of the variants [38] is an example of an informative approach.…”