Heterostyly.

Scharman and Lenhard introduce heterostyly, a phenomenon where individuals in a plant population produce flowers with more than one morphologically distinct form.

The gain-of-function mutation blf13 in the barley orthologue of the rice growth regulator NARROW LEAF1 is associated with increased leaf width.

Canopy architecture in cereals plays an important role in determining yield. Leaf width represents one key aspect of this canopy architecture. However, our understanding of leaf width control in cereals remains incomplete. Classical mutagenesis studies in barely identified multiple morphological mutants, including those with differing leaf widths. Of these, we characterized the broad leaf13 (blf13) mutant in detail. Mutant plants form wider leaves due to increased post-initiation growth and cell proliferation. The mutant phenotype perfectly co-segregated with a missense mutation in the HvHNT1 gene which affected a highly conserved region of the encoded protein, orthologous to the rice NARROW LEAF1 (NAL1) protein. Causality of this mutation for the blf13 phenotype is further supported by correlative transcriptomic analyses and protein-protein interaction studies showing that the mutant HvNHT1 protein interacts more strongly with a known interactor than wild-type HvHNT1. The mutant HvHNT1 protein also showed stronger homodimerization compared with wild-type HvHNT1, and homology modelling suggested an additional interaction site between HvHNT1 monomers due to the blf13 mutation. Thus, the blf13 mutation parallels known gain-of-function NAL1 alleles in rice that increase leaf width and grain yield, suggesting that the blf13 mutation may have a similar agronomic potential in barley.

Implementation of different relationship estimate methodologies in breeding value prediction in kiwiberry (Actinidia arguta).

In dioecious crops such as Actinidia arguta (kiwiberries), some of the main challenges when breeding for fruit characteristics are the selection of potential male parents and the long juvenile period. Currently, breeding values of male parents are estimated through progeny tests, which makes the breeding of new kiwiberry cultivars time-consuming and costly. The application of best linear unbiased prediction (BLUP) would allow direct estimation of sex-related traits and speed up kiwiberry breeding. In this study, we used a linear mixed model approach to estimate narrow sense heritability for one vine-related trait and five fruit-related traits for two incomplete factorial crossing designs. We obtained BLUPs for all genotypes, taking into consideration whether the relationship was pedigree-based or marker-based. Owing to the high cost of genome sequencing, it is important to understand the effects of different sources of relationship matrices on estimating breeding values across a breeding population. Because of the increasing implementation of genomic selection in crop breeding, we compared the effects of incorporating different sources of information in building relationship matrices and ploidy levels on the accuracy of BLUPs' heritability and predictive ability. As kiwiberries are autotetraploids, multivalent chromosome formation and occasionally double reduction can occur during meiosis, and this can affect the accuracy of prediction. This study innovates the breeding programme of autotetraploid kiwiberries. We demonstrate that the accuracy of BLUPs of male siblings, without phenotypic observations, strongly improved when a tetraploid marker-based relationship matrix was used rather than parental BLUPs and female siblings with phenotypic observations. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01419-8.

Testing the effect of individual scent compounds on pollinator attraction in nature using quasi-isogenic Capsella lines.

PREMISE: Floral scent, usually consisting of multiple compounds, is a complex trait, and its role in pollinator attraction has received increasing attention. However, disentangling the effect of individual floral scent compounds is difficult due to the complexity of isolating the effect of single compounds by traditional methods. METHODS: Using available quasi-isogenic lines (qILs) that were generated as part of the original mapping of the floral scent volatile-related loci CNL1 (benzaldehyde) and TPS2 (ß-ocimene) in Capsella, we generated four genotypes that should only differ in these two compounds. Plants of the four genotypes were introduced into a common garden outside the natural range of C. rubella or C. grandiflora, with individuals of a self-compatible C. grandiflora line as pollen donors, whose different genetic background facilitates the detection of outcrossing events. Visitors to flowers of all five genotypes were compared, and the seeds set during the common-garden period were collected for high-throughput amplicon-based sequencing to estimate their outcrossing rates. RESULTS: Benzaldehyde and ß-ocimene emissions were detected in the floral scent of corresponding genotypes. While some pollinator groups showed specific visitation preferences depending on scent compounds, the outcrossing rates in seeds did not vary among the four scent-manipulated genotypes. CONCLUSIONS: The scent-manipulated Capsella materials constructed using qILs provide a powerful system to study the ecological effects of individual floral scent compounds under largely natural environments. In Capsella, individual benzaldehyde and ß-ocimene emission may act as attractants for different types of pollinators.

Correction: Suppression of class I compensated cell enlargement by xs2 mutation is mediated by salicylic acid signaling.

[This corrects the article DOI: 10.1371/journal.pgen.1008873.].

Genomic and epigenomic determinants of heat stress-induced transcriptional memory in Arabidopsis.

BACKGROUND: Transcriptional regulation is a key aspect of environmental stress responses. Heat stress induces transcriptional memory, i.e., sustained induction or enhanced re-induction of transcription, that allows plants to respond more efficiently to a recurrent HS. In light of more frequent temperature extremes due to climate change, improving heat tolerance in crop plants is an important breeding goal. However, not all heat stress-inducible genes show transcriptional memory, and it is unclear what distinguishes memory from non-memory genes. To address this issue and understand the genome and epigenome architecture of transcriptional memory after heat stress, we identify the global target genes of two key memory heat shock transcription factors, HSFA2 and HSFA3, using time course ChIP-seq. RESULTS: HSFA2 and HSFA3 show near identical binding patterns. In vitro and in vivo binding strength is highly correlated, indicating the importance of DNA sequence elements. In particular, genes with transcriptional memory are strongly enriched for a tripartite heat shock element, and are hallmarked by several features: low expression levels in the absence of heat stress, accessible chromatin environment, and heat stress-induced enrichment of H3K4 trimethylation. These results are confirmed by an orthogonal transcriptomic data set using both de novo clustering and an established definition of memory genes. CONCLUSIONS: Our findings provide an integrated view of HSF-dependent transcriptional memory and shed light on its sequence and chromatin determinants, enabling the prediction and engineering of genes with transcriptional memory behavior.

Pollen-stigma incompatibility within and between species: Tread lightly, sedate the dogs, and don't wake the guards!

In a recent issue of Nature, Huang et al. identify and show how to overcome the barriers to successful pollen germination after interspecific crosses.1 Their findings answer a long-standing question about reproductive barriers in flowering plants and open the door to harnessing genetic diversity of distant relatives for crop improvement.

Poly(A) polymerase 1 contributes to competence acquisition of pollen tubes growing through the style in Arabidopsis thaliana.

Polyadenylation of mRNAs is critical for their export from the nucleus, stability, and efficient translation. The Arabidopsis thaliana genome encodes three isoforms of canonical nuclear poly(A) polymerase (PAPS) that redundantly polyadenylate the bulk of pre-mRNAs. However, previous studies have indicated that subsets of pre-mRNAs are preferentially polyadenylated by either PAPS1 or the other two isoforms. Such functional specialization raises the possibility of an additional level of gene-expression control in plants. Here we test this notion by studying the function of PAPS1 in pollen-tube growth and guidance. Pollen tubes growing through female tissue acquire the competence to find ovules efficiently and upregulate PAPS1 expression at the transcriptional, but not detectably at the protein level compared with in vitro grown pollen tubes. Using the temperature-sensitive paps1-1 allele we show that PAPS1 activity during pollen-tube growth is required for full acquisition of competence, resulting in inefficient fertilization by paps1-1 mutant pollen tubes. While these mutant pollen tubes grow almost at the wild-type rate, they are compromised in locating the micropyles of ovules. Previously identified competence-associated genes are less expressed in paps1-1 mutant than in wild-type pollen tubes. Estimating the poly(A) tail lengths of transcripts suggests that polyadenylation by PAPS1 is associated with reduced transcript abundance. Our results therefore suggest that PAPS1 plays a key role in the acquisition of competence and underline the importance of functional specialization between PAPS isoforms throughout different developmental stages.

Comparative Genomics Elucidates the Origin of a Supergene Controlling Floral Heteromorphism.

Supergenes are nonrecombining genomic regions ensuring the coinheritance of multiple, coadapted genes. Despite the importance of supergenes in adaptation, little is known on how they originate. A classic example of supergene is the S locus controlling heterostyly, a floral heteromorphism occurring in 28 angiosperm families. In Primula, heterostyly is characterized by the cooccurrence of two complementary, self-incompatible floral morphs and is controlled by five genes clustered in the hemizygous, ca. 300-kb S locus. Here, we present the first chromosome-scale genome assembly of any heterostylous species, that of Primula veris (cowslip). By leveraging the high contiguity of the P. veris assembly and comparative genomic analyses, we demonstrated that the S-locus evolved via multiple, asynchronous gene duplications and independent gene translocations. Furthermore, we discovered a new whole-genome duplication in Ericales that is specific to the Primula lineage. We also propose a mechanism for the origin of S-locus hemizygosity via nonhomologous recombination involving the newly discovered two pairs of CFB genes flanking the S locus. Finally, we detected only weak signatures of degeneration in the S locus, as predicted for hemizygous supergenes. The present study provides a useful resource for future research addressing key questions on the evolution of supergenes in general and the S locus in particular: How do supergenes arise? What is the role of genome architecture in the evolution of complex adaptations? Is the molecular architecture of heterostyly supergenes across angiosperms similar to that of Primula?

Female self-incompatibility type in heterostylous Primula is determined by the brassinosteroid-inactivating cytochrome P450 CYP734A50.

Most flowering plants are hermaphrodites, with flowers having both male and female reproductive organs. One widespread adaptation to limit self-fertilization is self-incompatibility (SI), where self-pollen fails to fertilize ovules.1,2 In homomorphic SI, many morphologically indistinguishable mating types are found, although in heteromorphic SI, the two or three mating types are associated with different floral morphologies.3-6 In heterostylous Primula, a hemizygous supergene determines a short-styled S-morph and a long-styled L-morph, corresponding to two different mating types, and full seed set only results from intermorph crosses.7-9 Style length is controlled by the brassinosteroid (BR)-inactivating cytochrome P450 CYP734A50,10 yet it remains unclear what defines the male and female incompatibility types. Here, we show that CYP734A50 also determines the female incompatibility type. Inactivating CYP734A50 converts short S-morph styles into long styles with the same incompatibility behavior as L-morph styles, and this effect can be mimicked by exogenous BR treatment. In vitro responses of S- and L-morph pollen grains and pollen tubes to increasing BR levels could only partly explain their different in vivo behavior, suggesting both direct and indirect effects of the different BR levels in S- versus L-morph stigmas and styles in controlling pollen performance. This BR-mediated SI provides a novel mechanism for preventing self-fertilization. The joint control of morphology and SI by CYP734A50 has important implications for the evolutionary buildup of the heterostylous syndrome and provides a straightforward explanation for why essentially all of the derived self-compatible homostylous Primula species are long homostyles.11.

Phylogeography of a widely distributed plant species reveals cryptic genetic lineages with parallel phenotypic responses to warming and drought conditions.

To predict how widely distributed species will perform under future climate change, it is crucial to understand and reveal their underlying phylogenetics. However, detailed information about plant adaptation and its genetic basis and history remains scarce and especially widely distributed species receive little attention despite their putatively high adaptability. To examine the adaptation potential of a widely distributed species, we sampled the model plant Silene vulgaris across Europe. In a greenhouse experiment, we exposed the offspring of these populations to a climate change scenario for central Europe and revealed the population structure through whole-genome sequencing. Plants were grown under two temperatures (18°C and 21°C) and three precipitation regimes (65, 75, and 90 mm) to measure their response in biomass and fecundity-related traits. To reveal the population genetic structure, ddRAD sequencing was employed for a whole-genome approach. We found three major genetic clusters in S. vulgaris from Europe: one cluster comprising Southern European populations, one cluster of Western European populations, and another cluster containing central European populations. Population genetic diversity decreased with increasing latitude, and a Mantel test revealed significant correlations between F ST and geographic distances as well as between genetic and environmental distances. Our trait analysis showed that the genetic clusters significantly differed in biomass-related traits and in the days to flowering. However, half of the traits showed parallel response patterns to the experimental climate change scenario. Due to the differentiated but parallel response patterns, we assume that phenotypic plasticity plays an important role for the adaptation of the widely distributed species S. vulgaris and its intraspecific genetic lineages.

Multi-omics approach reveals the contribution of KLU to leaf longevity and drought tolerance.

KLU, encoded by a cytochrome P450 CYP78A family gene, generates an important-albeit unknown-mobile signal that is distinct from the classical phytohormones. Multiple lines of evidence suggest that KLU/KLU-dependent signaling functions in several vital developmental programs, including leaf initiation, leaf/floral organ growth, and megasporocyte cell fate. However, the interactions between KLU/KLU-dependent signaling and the other classical phytohormones, as well as how KLU influences plant physiological responses, remain poorly understood. Here, we applied in-depth, multi-omics analysis to monitor transcriptome and metabolome dynamics in klu-mutant and KLU-overexpressing Arabidopsis plants. By integrating transcriptome sequencing data and primary metabolite profiling alongside phytohormone measurements, our results showed that cytokinin signaling, with its well-established function in delaying leaf senescence, was activated in KLU-overexpressing plants. Consistently, KLU-overexpressing plants exhibited significantly delayed leaf senescence and increased leaf longevity, whereas the klu-mutant plants showed early leaf senescence. In addition, proline biosynthesis and catabolism were enhanced following KLU overexpression owing to increased expression of genes associated with proline metabolism. Furthermore, KLU-overexpressing plants showed enhanced drought-stress tolerance and reduced water loss. Collectively, our work illustrates a role for KLU in positively regulating leaf longevity and drought tolerance by synergistically activating cytokinin signaling and promoting proline metabolism. These data promote KLU as a potential ideal genetic target to improve plant fitness.

Fairy circles in Namibia are assembled from genetically distinct grasses.

Fairy circles are striking regularly sized and spaced, bare circles surrounded by Stipagrostis grasses that occur over thousands of square kilometres in Namibia. The mechanisms explaining their origin, shape, persistence and regularity remain controversial. One hypothesis for the formation of vegetation rings is based on the centrifugal expansion of a single individual grass plant, via clonal growth and die-back in the centre. Clonality could explain FC origin, shape and long-term persistence as well as their regularity, if one clone competes with adjacent clones. Here, we show that for virtually all tested fairy circles the periphery is not exclusively made up of genetically identical grasses, but these peripheral grasses belong to more than one unrelated genet. These results do not support a clonal explanation for fairy circles. Lack of clonality implies that a biological reason for their origin, shape and regularity must emerge from competition between near neighbor individuals within each fairy circle. Such lack of clonality also suggests a mismatch between longevity of fairy circles versus their constituent plants. Furthermore, our findings of lack of clonality have implications for some models of spatial patterning of fairy circles that are based on self-organization.

Supergene evolution via stepwise duplications and neofunctionalization of a floral-organ identity gene.

Heterostyly represents a fascinating adaptation to promote outbreeding in plants that evolved multiple times independently. While l-morph individuals form flowers with long styles, short anthers, and small pollen grains, S-morph individuals have flowers with short styles, long anthers, and large pollen grains. The difference between the morphs is controlled by an S-locus "supergene" consisting of several distinct genes that determine different traits of the syndrome and are held together, because recombination between them is suppressed. In Primula, the S locus is a roughly 300-kb hemizygous region containing five predicted genes. However, with one exception, their roles remain unclear, as does the evolutionary buildup of the S locus. Here we demonstrate that the MADS-box GLOBOSA2 (GLO2) gene at the S locus determines anther position. In Primula forbesii S-morph plants, GLO2 promotes growth by cell expansion in the fused tube of petals and stamen filaments beneath the anther insertion point; by contrast, neither pollen size nor male incompatibility is affected by GLO2 activity. The paralogue GLO1, from which GLO2 arose by duplication, has maintained the ancestral B-class function in specifying petal and stamen identity, indicating that GLO2 underwent neofunctionalization, likely at the level of the encoded protein. Genetic mapping and phylogenetic analysis indicate that the duplications giving rise to the style-length-determining gene CYP734A50 and to GLO2 occurred sequentially, with the CYP734A50 duplication likely the first. Together these results provide the most detailed insight into the assembly of a plant supergene yet and have important implications for the evolution of heterostyly.

Establishment of the Embryonic Shoot Meristem Involves Activation of Two Classes of Genes with Opposing Functions for Meristem Activities.

The shoot meristem, a stem-cell-containing tissue initiated during plant embryogenesis, is responsible for continuous shoot organ production in postembryonic development. Although key regulatory factors including KNOX genes are responsible for stem cell maintenance in the shoot meristem, how the onset of such factors is regulated during embryogenesis is elusive. Here, we present evidence that the two KNOX genes STM and KNAT6 together with the two other regulatory genes BLR and LAS are functionally important downstream genes of CUC1 and CUC2, which are a redundant pair of genes that specify the embryonic shoot organ boundary. Combined expression of STM with any of KNAT6, BLR, and LAS can efficiently rescue the defects of shoot meristem formation and/or separation of cotyledons in cuc1cuc2 double mutants. In addition, CUC1 and CUC2 are also required for the activation of KLU, a cytochrome P450-encoding gene known to restrict organ production, and KLU counteracts STM in the promotion of meristem activity, providing a possible balancing mechanism for shoot meristem maintenance. Together, these results establish the roles for CUC1 and CUC2 in coordinating the activation of two classes of genes with opposite effects on shoot meristem activity.

Adaptive reduction of male gamete number in the selfing plant Arabidopsis thaliana.

The number of male gametes is critical for reproductive success and varies between and within species. The evolutionary reduction of the number of pollen grains encompassing the male gametes is widespread in selfing plants. Here, we employ genome-wide association study (GWAS) to identify underlying loci and to assess the molecular signatures of selection on pollen number-associated loci in the predominantly selfing plant Arabidopsis thaliana. Regions of strong association with pollen number are enriched for signatures of selection, indicating polygenic selection. We isolate the gene REDUCED POLLEN NUMBER1 (RDP1) at the locus with the strongest association. We validate its effect using a quantitative complementation test with CRISPR/Cas9-generated null mutants in nonstandard wild accessions. In contrast to pleiotropic null mutants, only pollen numbers are significantly affected by natural allelic variants. These data support theoretical predictions that reduced investment in male gametes is advantageous in predominantly selfing species.

Suppression of class I compensated cell enlargement by xs2 mutation is mediated by salicylic acid signaling.

The regulation of leaf size has been studied for decades. Enhancement of post-mitotic cell expansion triggered by impaired cell proliferation in Arabidopsis is an important process for leaf size regulation, and is known as compensation. This suggests a key interaction between cell proliferation and cell expansion during leaf development. Several studies have highlighted the impact of this integration mechanism on leaf size determination; however, the molecular basis of compensation remains largely unknown. Previously, we identified extra-small sisters (xs) mutants which can suppress compensated cell enlargement (CCE) via a specific defect in cell expansion within the compensation-exhibiting mutant, angustifolia3 (an3). Here we revealed that one of the xs mutants, namely xs2, can suppress CCE not only in an3 but also in other compensation-exhibiting mutants erecta (er) and fugu2. Molecular cloning of XS2 identified a deleterious mutation in CATION CALCIUM EXCHANGER 4 (CCX4). Phytohormone measurement and expression analysis revealed that xs2 shows hyper activation of the salicylic acid (SA) response pathway, where activation of SA response can suppress CCE in compensation mutants. All together, these results highlight the regulatory connection which coordinates compensation and SA response.

Mapping-by-Sequencing via MutMap Identifies a Mutation in ZmCLE7 Underlying Fasciation in a Newly Developed EMS Mutant Population in an Elite Tropical Maize Inbred.

Induced point mutations are important genetic resources for their ability to create hypo- and hypermorphic alleles that are useful for understanding gene functions and breeding. However, such mutant populations have only been developed for a few temperate maize varieties, mainly B73 and W22, yet no tropical maize inbred lines have been mutagenized and made available to the public to date. We developed a novel Ethyl Methanesulfonate (EMS) induced mutation resource in maize comprising 2050 independent M2 mutant families in the elite tropical maize inbred ML10. By phenotypic screening, we showed that this population is of comparable quality with other mutagenized populations in maize. To illustrate the usefulness of this population for gene discovery, we performed rapid mapping-by-sequencing to clone a fasciated-ear mutant and identify a causal promoter deletion in ZmCLE7 (CLE7). Our mapping procedure does not require crossing to an unrelated parent, thus is suitable for mapping subtle traits and ones affected by heterosis. This first EMS population in tropical maize is expected to be very useful for the maize research community. Also, the EMS mutagenesis and rapid mapping-by-sequencing pipeline described here illustrate the power of performing forward genetics in diverse maize germplasms of choice, which can lead to novel gene discovery due to divergent genetic backgrounds.

Retracing the molecular basis and evolutionary history of the loss of benzaldehyde emission in the genus Capsella.

The transition from pollinator-mediated outbreeding to selfing has occurred many times in angiosperms. This is generally accompanied by a reduction in traits attracting pollinators, including reduced emission of floral scent. In Capsella, emission of benzaldehyde as a main component of floral scent has been lost in selfing C. rubella by mutation of cinnamate-CoA ligase CNL1. However, the biochemical basis and evolutionary history of this loss remain unknown, as does the reason for the absence of benzaldehyde emission in the independently derived selfer Capsella orientalis. We used plant transformation, in vitro enzyme assays, population genetics and quantitative genetics to address these questions. CNL1 has been inactivated twice independently by point mutations in C. rubella, causing a loss of enzymatic activity. Both inactive haplotypes are found within and outside of Greece, the centre of origin of C. rubella, indicating that they arose before its geographical spread. By contrast, the loss of benzaldehyde emission in C. orientalis is not due to an inactivating mutation in CNL1. CNL1 represents a hotspot for mutations that eliminate benzaldehyde emission, potentially reflecting the limited pleiotropy and large effect of its inactivation. Nevertheless, even closely related species have followed different evolutionary routes in reducing floral scent.