The Andromonoecious Sex Determination Gene Predates the Separation of Cucumis and Citrullus Genera.

Understanding the evolution of sex determination in plants requires the cloning and the characterization of sex determination genes. Monoecy is characterized by the presence of both male and female flowers on the same plant. Andromonoecy is characterized by plants carrying both male and bisexual flowers. In watermelon, the transition between these two sexual forms is controlled by the identity of the alleles at the A locus. We previously showed, in two Cucumis species, melon and cucumber, that the transition from monoecy to andromonoecy results from mutations in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, ACS-7/ACS2. To test whether the ACS-7/ACS2 function is conserved in cucurbits, we cloned and characterized ClACS7 in watermelon. We demonstrated co-segregation of ClACS7, the homolog of CmACS-7/CsACS2, with the A locus. Sequence analysis of ClACS7 in watermelon accessions identified three ClACS7 isoforms, two in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed and assayed the activity of the three protein isoforms. Like in melon and cucumber, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active ClACS7 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. ClACS7, like CmACS-7/CsACS2 in melon and cucumber, is highly expressed in carpel primordia of buds determined to develop carpels and not in male flowers. Based on this finding and previous investigations, we concluded that the monoecy gene, ACS7, likely predated the separation of the Cucumis and Citrullus genera.

A cucurbit androecy gene reveals how unisexual flowers develop and dioecy emerges.

Understanding the evolution of sex determination in plants requires identifying the mechanisms underlying the transition from monoecious plants, where male and female flowers coexist, to unisexual individuals found in dioecious species. We show that in melon and cucumber, the androecy gene controls female flower development and encodes a limiting enzyme of ethylene biosynthesis, ACS11. ACS11 is expressed in phloem cells connected to flowers programmed to become female, and ACS11 loss-of-function mutants lead to male plants (androecy). CmACS11 represses the expression of the male promoting gene CmWIP1 to control the development and the coexistence of male and female flowers in monoecious species. Because monoecy can lead to dioecy, we show how a combination of alleles of CmACS11 and CmWIP1 can create artificial dioecy.

AAA-ATPase FIDGETIN-LIKE 1 and Helicase FANCM Antagonize Meiotic Crossovers by Distinct Mechanisms.

Meiotic crossovers (COs) generate genetic diversity and are critical for the correct completion of meiosis in most species. Their occurrence is tightly constrained but the mechanisms underlying this limitation remain poorly understood. Here we identified the conserved AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) as a negative regulator of meiotic CO formation. We show that Arabidopsis FIGL1 limits CO formation genome-wide, that FIGL1 controls dynamics of the two conserved recombinases DMC1 and RAD51 and that FIGL1 hinders the interaction between homologous chromosomes, suggesting that FIGL1 counteracts DMC1/RAD51-mediated inter-homologue strand invasion to limit CO formation. Further, depleting both FIGL1 and the previously identified anti-CO helicase FANCM synergistically increases crossover frequency. Additionally, we showed that the effect of mutating FANCM on recombination is much lower in F1 hybrids contrasting from the phenotype of inbred lines, while figl1 mutation equally increases crossovers in both contexts. This shows that the modes of action of FIGL1 and FANCM are differently affected by genomic contexts. We propose that FIGL1 and FANCM represent two successive barriers to CO formation, one limiting strand invasion, the other disassembling D-loops to promote SDSA, which when both lifted, leads to a large increase of crossovers, without impairing meiotic progression.

Multiple mechanisms limit meiotic crossovers: TOP3α and two BLM homologs antagonize crossovers in parallel to FANCM.

Meiotic crossovers (COs) have two important roles, shuffling genetic information and ensuring proper chromosome segregation. Despite their importance and a large excess of precursors (i.e., DNA double-strand breaks, DSBs), the number of COs is tightly regulated, typically one to three per chromosome pair. The mechanisms ensuring that most DSBs are repaired as non-COs and the evolutionary forces imposing this constraint are poorly understood. Here we identified Topoisomerase3α (TOP3α) and the RECQ4 helicases--the Arabidopsis slow growth suppressor 1 (Sgs1)/Bloom syndrome protein (BLM) homologs--as major barriers to meiotic CO formation. First, the characterization of a specific TOP3α mutant allele revealed that, in addition to its role in DNA repair, this topoisomerase antagonizes CO formation. Further, we found that RECQ4A and RECQ4B constitute the strongest meiotic anti-CO activity identified to date, their concomitant depletion leading to a sixfold increase in CO frequency. In both top3α and recq4ab mutants, DSB number is unaffected, and extra COs arise from a normally minor pathway. Finally, both TOP3α and RECQ4A/B act independently of the previously identified anti-CO Fanconi anemia of complementation group M (FANCM) helicase. This finding shows that several parallel pathways actively limit CO formation and suggests that the RECQA/B and FANCM helicases prevent COs by processing different substrates. Despite a ninefold increase in CO frequency, chromosome segregation was unaffected. This finding supports the idea that CO number is restricted not because of mechanical constraints but likely because of the long-term costs of recombination. Furthermore, this work demonstrates how manipulating a few genes holds great promise for increasing recombination frequency in plant-breeding programs.

The kinesin AtPSS1 promotes synapsis and is required for proper crossover distribution in meiosis.

Meiotic crossovers (COs) shape genetic diversity by mixing homologous chromosomes at each generation. CO distribution is a highly regulated process. CO assurance forces the occurrence of at least one obligatory CO per chromosome pair, CO homeostasis smoothes out the number of COs when faced with variation in precursor number and CO interference keeps multiple COs away from each other along a chromosome. In several organisms, it has been shown that cytoskeleton forces are transduced to the meiotic nucleus via KASH- and SUN-domain proteins, to promote chromosome synapsis and recombination. Here we show that the Arabidopsis kinesin AtPSS1 plays a major role in chromosome synapsis and regulation of CO distribution. In Atpss1 meiotic cells, chromosome axes and DNA double strand breaks (DSBs) appear to form normally but only a variable portion of the genome synapses and is competent for CO formation. Some chromosomes fail to form the obligatory CO, while there is an increased CO density in competent regions. However, the total number of COs per cell is unaffected. We further show that the kinesin motor domain of AtPSS1 is required for its meiotic function, and that AtPSS1 interacts directly with WIP1 and WIP2, two KASH-domain proteins. Finally, meiocytes missing AtPSS1 and/or SUN proteins show similar meiotic defects suggesting that AtPSS1 and SUNs act in the same pathway. This suggests that forces produced by the AtPSS1 kinesin and transduced by WIPs/SUNs, are required to authorize complete synapsis and regulate maturation of recombination intermediates into COs. We suggest that a form of homeostasis applies, which maintains the total number of COs per cell even if only a part of the genome is competent for CO formation.

FANCM-associated proteins MHF1 and MHF2, but not the other Fanconi anemia factors, limit meiotic crossovers.

Genetic recombination is important for generating diversity and to ensure faithful segregation of chromosomes at meiosis. However, few crossovers (COs) are formed per meiosis despite an excess of DNA double-strand break precursors. This reflects the existence of active mechanisms that limit CO formation. We previously showed that AtFANCM is a meiotic anti-CO factor. The same genetic screen now identified AtMHF2 as another player of the same anti-CO pathway. FANCM and MHF2 are both Fanconi Anemia (FA) associated proteins, prompting us to test the other FA genes conserved in Arabidopsis for a role in CO control at meiosis. This revealed that among the FA proteins tested, only FANCM and its two DNA-binding co-factors MHF1 and MHF2 limit CO formation at meiosis.

The Arabidopsis HEI10 is a new ZMM protein related to Zip3.

In numerous species, the formation of meiotic crossovers is largely under the control of a group of proteins known as ZMM. Here, we identified a new ZMM protein, HEI10, a RING finger-containing protein that is well conserved among species. We show that HEI10 is structurally and functionally related to the yeast Zip3 ZMM and that it is absolutely required for class I crossover (CO) formation in Arabidopsis thaliana. Furthermore, we show that it is present as numerous foci on the chromosome axes and the synaptonemal complex central element until pachytene. Then, from pachytene to diakinesis, HEI10 is retained at a limited number of sites that correspond to class I COs, where it co-localises with MLH1. Assuming that HEI10 early staining represents an early selection of recombination intermediates to be channelled into the ZMM pathway, HEI10 would therefore draw a continuity between early chosen recombination intermediates and final class I COs.