A WOX homolog disrupted by a transposon led to the loss of spines and contributed to the domestication of lettuce.

The loss of spines is one of the most important domestication traits for lettuce (Lactuca sativa). However, the genetics and regulation of spine development in lettuce remain unclear. We examined the genetics of spines in lettuce using a segregating population derived from a cross between cultivated and wild lettuce (Lactuca serriola). A gene encoding WUSCHEL-related homeobox transcription factor, named as WOX-SPINE1 (WS1), was identified as the candidate gene controlling the spine development in lettuce, and its function on spines was verified. A CACTA transposon was found to be inserted into the first exon of the ws1 allele, knocking out its function and leading to the lack of spines in cultivated lettuce. All lettuce cultivars investigated have the nonfunctional ws1 gene, and a selection sweep was found at the WS1 locus, suggesting its important role in lettuce domestication. The expression levels of WS1 were associated with the density of spines among different accessions of wild lettuce. At least two independent loss-of-function mutations in the ws1 gene caused the loss of spines in wild lettuce. These findings provide new insights into the development of spines and facilitate the exploitation of wild genetic resources in future lettuce breeding programs.

Seed color in lettuce is determined by the LsTT2, LsCHS, and Ls2OGD genes from the flavonoid biosynthesis pathway.

KEY MESSAGE: The mutated LsTT2 and Ls2OGD genes are responsible for white seeds and yellow seeds in lettuce, respectively. Three LsCHS genes are involved in the biosynthesis of flavonoid in seed coats. Lettuce seeds have several different colors, including black, yellow, and white. The genetic mechanisms underlying color variations of lettuce seeds remain unknown. We used genome-wide association studies (GWAS) and map-based cloning approaches to clone genes controlling the color of lettuce seeds. LsTT2, which encodes an R2R3-MYB transcription factor and is homologous to the TT2 gene in Arabidopsis, was shown to be the causal gene for the variation of black and white seeds in lettuce. A point mutation leads to the lack of stop codon in the LsTT2 transcript, resulting in white seeds. Knockout of the LsTT2 gene converted black seeds to white seeds. The locus controlling yellow seeds was mapped to Chromosome 2. Knockout of two 2-oxoglutarate-dependent dioxygenases (2OGD) genes from the candidate region converted black seeds to yellow seeds, suggesting that these two 2OGD proteins catalyze the conversion of yellow metabolites to black metabolites. We also showed that three LsCHS genes from the candidate region are associated with flavonoid biosynthesis in seeds. Knockout mutants of the three LsCHS genes decreased color intensity. This study provides new insights into the regulation of flavonoid biosynthesis in plants.

Loss-of-function of SAWTOOTH 1 affects leaf dorsiventrality genes to promote leafy heads in lettuce.

The mechanisms underlying leafy heads in vegetables are poorly understood. Here, we cloned a quantitative trait locus (QTL) controlling leafy heads in lettuce (Lactuca sativa). The QTL encodes a transcription factor, SAWTOOTH 1 (LsSAW1), which has a BEL1-like homeodomain and is a homolog of Arabidopsis thaliana. A 1-bp deletion in Lssaw1 contributes to the development of leafy heads. Laser-capture microdissection and RNA-sequencing showed that LsSAW1 regulates leaf dorsiventrality and loss-of-function of Lssaw1 downregulates the expression of many adaxial genes but upregulates abaxial genes. LsSAW1 binds to the promoter region of the adaxial gene ASYMMETRIC LEAVES 1 (LsAS1) to upregulate its expression. Overexpression of LsAS1 compromised the effects of Lssaw1 on heading. LsSAW1 also binds to the promoter region of the abaxial gene YABBY 1 (LsYAB1), but downregulates its expression. Overexpression of LsYAB1 led to bending leaves in LsSAW1 genotypes. LsSAW1 directly interacts with KNOTTED 1 (LsKN1), which is necessary for leafy heads in lettuce. RNA-seq data showed that LsSAW1 and LsKN1 exert antagonistic effects on the expression of thousands of genes. LsSAW1 compromises the ability of LsKN1 to repress LsAS1. Our results suggest that downregulation or loss-of-function of adaxial genes and upregulation of abaxial genes allow for the development of leafy heads.

Up-regulation of LsKN1 promotes cytokinin and suppresses gibberellin biosynthesis to generate wavy leaves in lettuce.

Lettuce (Lactuca sativa) is one of the most popular vegetables worldwide, and diverse leaf shapes, including wavy leaves, are important commercial traits. In this study, we examined the genetics of wavy leaves using an F2 segregating population, and identified a major QTL controlling wavy leaves. The candidate region contained LsKN1, which has previously been shown to be indispensable for leafy heads in lettuce. Complementation tests and knockout experiments verified the function of LsKN1 in producing wavy leaves. The LsKN1∇ allele, which has the insertion of a transposon and has previously been shown to control leafy heads, promoted wavy leaves in our population. Transposition of the CACTA transposon from LsKN1 compromised its function for wavy leaves. High expression of LsKN1 up-regulated several key genes associated with cytokinin (CK) to increase the content in the leaves, whereas it down-regulated the expression of genes in the gibberellin (GA) biosynthesis pathway to decrease the content. Application of CK to leaves enhanced the wavy phenotype, while application of GA dramatically flattened the leaves. We conclude that the changes in CK and GA contents that result from high expression of LsKN1 switch determinate cells to indeterminate, and consequently leads to the development of wavy leaves.

LsNRL4 enhances photosynthesis and decreases leaf angles in lettuce.

Lettuce (Lactuca sativa) is one of the most important vegetables worldwide and an ideal plant for producing protein drugs. Both well-functioning chloroplasts that perform robust photosynthesis and small leaf angles that enable dense planting are essential for high yields. In this study, we used an F2 population derived from a cross between a lettuce cultivar with pale-green leaves and large leaf angles to a cultivar with dark-green leaves and small leaf angles to clone LsNRL4, which encodes an NPH3/RPT2-Like (NRL) protein. Unlike other NRL proteins in lettuce, the LsNRL4 lacks the BTB domain. Knockout mutants engineered using CRISPR/Cas9 and transgenic lines overexpressing LsNRL4 verified that LsNRL4 contributes to chloroplast development, photosynthesis and leaf angle. The LsNRL4 gene was not present in the parent with pale-green leaves and enlarged leaf angles. Loss of LsNRL4 results in the enlargement of chloroplasts, decreases in the amount of cellular space allocated to chloroplasts and defects in secondary cell wall biosynthesis in lamina joints. Overexpressing LsNRL4 significantly improved photosynthesis and decreased leaf angles. Indeed, the plant architecture of the overexpressing lines is ideal for dense planting. In summary, we identified a novel NRL gene that enhances photosynthesis and influences plant architecture. Our study provides new approaches for the breeding of lettuce that can be grown in dense planting in the open field or in modern plant factories. LsNRL4 homologues may also be used in other crops to increase photosynthesis and improve plant architecture.

The upregulated LsKN1 gene transforms pinnately to palmately lobed leaves through auxin, gibberellin, and leaf dorsiventrality pathways in lettuce.

Leaf shape represents a vital agronomic trait for leafy vegetables such as lettuce. Some lettuce cultivars produce lobed leaves, varying from pinnately to palmately lobed, but the genetic mechanisms remain unclear. In this study, we cloned one major quantitative trait locus (QTL) controlling palmately lobed leaves. The candidate gene, LsKN1, encodes a homeobox transcription factor, and has been shown previously to be critical for the development of leafy heads in lettuce. The LsKN1 allele that is upregulated by the insertion of a transposon promotes the development of palmately lobed leaves. We demonstrated that LsKN1 upregulated LsCUC2 and LsCUC3 through different mechanisms, and their upregulation was critical for the development of palmately lobed leaves. LsKN1 binds the promoter of LsPID to promote auxin biosynthesis, which positively contributes to the development of palmately lobed leaves. In contrast, LsKN1 suppresses GA biosynthesis to promote palmately lobed leaves. LsKN1 also binds to the promoter of LsAS1, a dorsiventrality gene, to downregulate its expression. Overexpression of the LsAS1 gene compromised the effects of the LsKN1 gene changing palmately to pinnately lobed leaves. Our study illustrated that the upregulated LsKN1 gene led to palmately lobed leaves in lettuce by integrating several downstream pathways, including auxin, gibberellin, and leaf dorsiventrality pathways.

Alternative splicing triggered by the insertion of a CACTA transposon attenuates LsGLK and leads to the development of pale-green leaves in lettuce.

Lettuce (Lactuca sativa) is one of the most important vegetable crops in the world. As a leafy vegetable, the polymorphism of lettuce leaves from dark to pale green is an important trait. However, the genetic and molecular mechanisms underlying such variations remain poorly understood. In this study, one major locus controlling the polymorphism of dark- and pale-green leaves in lettuce was identified using genome-wide association studies (GWAS). This locus was then fine mapped to an interval of 5375 bp on chromosome 4 using a segregating population containing 2480 progeny. Only one gene, homologous to the GLK genes in Arabidopsis and other plants, is present in the candidate region. A complementation test confirmed that the candidate gene, LsGLK, contributes to the variation of dark- and pale-green leaves. Sequence analysis showed that a CACTA transposon of 7434 bp was inserted 10 bp downstream of the stop codon of LsGLK, followed by a duplication of a 1826-bp fragment covering exons 3-6 of the LsGLK gene. The transposon insertion did not change the expression level of the LsGLK gene. However, because of alternative splicing, only 6% of the transcripts produced from the transposon insertion were wild-type transcripts, which led to the production of pale-green leaves. An evolutionary analysis revealed that the insertion of the CACTA transposon occurred in cultivated lettuce and might have been selected in particular cultivars to satisfy the diverse demands of consumers. In this study, we demonstrated that a transposon insertion near a gene may affect its splicing and consequently generate phenotypic variations.

Two TOBAMOVIRUS MULTIPLICATION 2A homologs in tobacco control asymptomatic response to tobacco mosaic virus.

The most common response of a host to pathogens is arguably the asymptomatic response. However, the genetic and molecular mechanisms responsible for asymptomatic responses to pathogens are poorly understood. Here we report on the genetic cloning of two genes controlling the asymptomatic response to tobacco mosaic virus (TMV) in cultivated tobacco (Nicotiana tabacum). These two genes are homologous to tobamovirus multiplication 2A (TOM2A) from Arabidopsis, which was shown to be critical for the accumulation of TMV. Expression analysis indicates that the TOM2A genes might play fundamental roles in plant development or in responses to stresses. Consistent with this hypothesis, a null allele of the TOM2A ortholog in tomato (Solanum lycopersicum) led to the development of bent branches and a high tolerance to both TMV and tomato mosaic virus (ToMV). However, the TOM2A ortholog in Nicotiana glauca did not account for the asymptomatic response to TMV in N. glauca. We showed that TOM2A family is plant-specific and originated from Chlorophyte, and the biological functions of TOM2A orthologs to promote TMV accumulation are highly conserved in the plant kingdom-in both TMV host and nonhost species. In addition, we showed that the interaction between tobacco TOM1 and TOM2A orthologs in plant species is conserved, suggesting a conserved nature of TOM1-TOM2A module in promoting TMV multiplication in plants. The tradeoff between host development, the resistance of hosts to pathogens, and their influence on gene evolution are discussed. Our results shed light on mechanisms that contribute to asymptomatic responses to viruses in plants and provide approaches for developing TMV/ToMV-resistant crops.

Frequent gain- and loss-of-function mutations of the BjMYB113 gene accounted for leaf color variation in Brassica juncea.

BACKGROUND: Mustard (Brassica juncea) is an important economic vegetable, and some cultivars have purple leaves and accumulate more anthocyanins than the green. The genetic and evolution of purple trait in mustard has not been well studied. RESULT: In this study, free-hand sections and metabolomics showed that the purple leaves of mustard accumulated more anthocyanins than green ones. The gene controlling purple leaves in mustard, Mustard Purple Leaves (MPL), was genetically mapped and a MYB113-like homolog was identified as the candidate gene. We identified three alleles of the MYB113-like gene, BjMYB113a from a purple cultivar, BjMYB113b and BjMYB113c from green cultivars. A total of 45 single nucleotide polymorphisms (SNPs) and 8 InDels were found between the promoter sequences of the purple allele BjMYB113a and the green allele BjMYB113b. On the other hand, the only sequence variation between the purple allele BjMYB113a and the green allele BjMYB113c is an insertion of 1,033-bp fragment in the 3'region of BjMYB113c. Transgenic assay and promoter activity studies showed that the polymorphism in the promoter region was responsible for the up-regulation of the purple allele BjMYB113a and high accumulation of anthocyanin in the purple cultivar. The up-regulation of BjMYB113a increased the expression of genes in the anthocyanin biosynthesis pathway including BjCHS, BjF3H, BjF3'H, BjDFR, BjANS and BjUGFT, and consequently led to high accumulation of anthocyanin. However, the up-regulation of BjMYB113 was compromised by the insertion of 1,033-bp in 3'region of the allele BjMYB113c. CONCLUSIONS: Our results contribute to a better understanding of the genetics and evolution of the BjMYB113 gene controlling purple leaves and provide useful information for further breeding programs of mustard.

Whole-genome resequencing of 445 Lactuca accessions reveals the domestication history of cultivated lettuce.

Lettuce (Lactuca sativa) is an important vegetable crop worldwide. Cultivated lettuce is believed to be domesticated from L. serriola; however, its origins and domestication history remain to be elucidated. Here, we sequenced a total of 445 Lactuca accessions, including major lettuce crop types and wild relative species, and generated a comprehensive map of lettuce genome variations. In-depth analyses of population structure and demography revealed that lettuce was first domesticated near the Caucasus, which was marked by loss of seed shattering. We also identified the genetic architecture of other domestication traits and wild introgressions in major resistance clusters in the lettuce genome. This study provides valuable genomic resources for crop breeding and sheds light on the domestication history of cultivated lettuce.

Genome sequence and evolution of Betula platyphylla.

Betula L. (birch) is a pioneer hardwood tree species with ecological, economic, and evolutionary importance in the Northern Hemisphere. We sequenced the Betula platyphylla genome and assembled the sequences into 14 chromosomes. The Betula genome lacks evidence of recent whole-genome duplication and has the same paleoploidy level as Vitis vinifera and Prunus mume. Phylogenetic analysis of lignin pathway genes coupled with tissue-specific expression patterns provided clues for understanding the formation of higher ratios of syringyl to guaiacyl lignin observed in Betula species. Our transcriptome analysis of leaf tissues under a time-series cold stress experiment revealed the presence of the MEKK1-MKK2-MPK4 cascade and six additional mitogen-activated protein kinases that can be linked to a gene regulatory network involving many transcription factors and cold tolerance genes. Our genomic and transcriptome analyses provide insight into the structures, features, and evolution of the B. platyphylla genome. The chromosome-level genome and gene resources of B. platyphylla obtained in this study will facilitate the identification of important and essential genes governing important traits of trees and genetic improvement of B. platyphylla.

Upregulation of a KN1 homolog by transposon insertion promotes leafy head development in lettuce.

Leafy head is a unique type of plant architecture found in some vegetable crops, with leaves bending inward to form a compact head. The genetic and molecular mechanisms underlying leafy head in vegetables remain poorly understood. We genetically fine-mapped and cloned a major quantitative trait locus controlling heading in lettuce. The candidate gene (LsKN1) is a homolog of knotted 1 (KN1) from Zea mays Complementation and CRISPR/Cas9 knockout experiments confirmed the role of LsKN1 in heading. In heading lettuce, there is a CACTA-like transposon inserted into the first exon of LsKN1 (LsKN1▽). The transposon sequences act as a promoter rather than an enhancer and drive high expression of LsKN1▽. The enhanced expression of LsKN1▽ is necessary but not sufficient for heading in lettuce. Data from ChIP-sequencing, electrophoretic mobility shift assays, and dual luciferase assays indicate that the LsKN1▽ protein binds the promoter of LsAS1 and down-regulates its expression to alter leaf dorsoventrality. This study provides insight into plant leaf development and will be useful for studies on heading in other vegetable crops.

Characterization of four polymorphic genes controlling red leaf colour in lettuce that have undergone disruptive selection since domestication.

Anthocyanins protect plants from biotic and abiotic stressors and provide great health benefits to consumers. In this study, we cloned four genes (Red Lettuce Leaves 1 to 4: RLL1 to RLL4) that contribute to colour variations in lettuce. The RLL1 gene encodes a bHLH transcription factor, and a 5-bp deletion in some cultivars abolishes its function to activate the anthocyanin biosynthesis pathway. The RLL2 gene encodes an R2R3-MYB transcription factor, which was derived from a duplication followed by mutations in its promoter region. The RLL3 gene encodes an R2-MYB transcription factor, which down-regulates anthocyanin biosynthesis through competing with RLL2 for interaction with RLL1; a mis-sense mutation compromises the capacity of RLL3 to bind RLL1. The RLL4 gene encodes a WD-40 transcription factor, homologous to the RUP genes suppressing the UV-B signal transduction pathway in Arabidopsis; a mis-sense mutation in rll4 attenuates its suppressing function, leading to a high concentration of anthocyanins. Sequence analysis of the RLL1-RLL4 genes from wild and cultivated lettuce showed that their function-changing mutations occurred after domestication. The mutations in rll1 disrupt anthocyanin biosynthesis, while the mutations in RLL2, rll3 and rll4 activate anthocyanin biosynthesis, showing disruptive selection for leaf colour during domestication of lettuce. The characterization of multiple polymorphic genes in this study provides the necessary molecular resources for the rational breeding of lettuce cultivars with distinct levels of red pigments and green cultivars with high levels of health-promoting flavonoids.

Independent activation of the BoMYB2 gene leading to purple traits in Brassica oleracea.

KEY MESSAGE: Transposon insertion and point mutation independently activated the BoMYB2 gene in three purple cultivars of Brassica oleracea including kale, kohlrabi, and cabbage. Several varieties of B. oleracea have both green and purple cultivars. In this study, the causal genes for the purple traits in kale, kohlrabi and cabbage were cloned using map-based cloning approach. The purple traits in all three varieties were mapped to the same locus as the BoMYB2 gene in cauliflower. Surprisingly, the insertion of Harbinger transposon of BoMYB2 in cauliflower was not found in purple kale, kohlrabi and cabbage. Sequencing of the BoMYB2 gene in purple kale and purple kohlrabi discovered a 7606 bp CACTA-like transposon in its promoter region. Transient assay and promoter activity study showed that the insertion upregulated the expression of the BoMYB2 gene. On the other hand, the activation of the BoMYB2 gene in purple cabbage was caused by point mutation and/or 1-bp insertion in its promoter region. Sequence analysis of the BoMYB2 gene in different varieties suggested that the activating events most likely occurred independently after the divergence of cabbage, cauliflower, and kale/kohlrabi. Our results not only contribute to a better understanding of anthocyanin inheritance in B. oleracea, but also provide useful information for future hybrid breeding of purple cultivars through combination of different functional alleles of the BoMYB2 gene.

RNA sequencing provides insights into the evolution of lettuce and the regulation of flavonoid biosynthesis.

Different horticultural types of lettuce exhibit tremendous morphological variation. However, the molecular basis for domestication and divergence among the different horticultural types of lettuce remains unknown. Here, we report the RNA sequencing of 240 lettuce accessions sampled from the major horticultural types and wild relatives, generating 1.1 million single-nucleotide polymorphisms (SNPs). Demographic modeling indicates that there was a single domestication event for lettuce. We identify a list of regions as putative selective sweeps that occurred during domestication and divergence, respectively. Genome-wide association studies (GWAS) identify 5311 expression quantitative trait loci (eQTL) regulating the expression of 4105 genes, including nine eQTLs regulating genes associated with flavonoid biosynthesis. GWAS for leaf color detects six candidate loci responsible for the variation of anthocyanins in lettuce leaves. Our study provides a comprehensive understanding of the domestication and the accumulation of anthocyanins in lettuce, and will facilitate the breeding of cultivars with improved nutritional value.

Molecular Modeling Studies of 11ß-Hydroxysteroid Dehydrogenase Type 1 Inhibitors through Receptor-Based 3D-QSAR and Molecular Dynamics Simulations.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is a potential target for the treatment of numerous human disorders, such as diabetes, obesity, and metabolic syndrome. In this work, molecular modeling studies combining molecular docking, 3D-QSAR, MESP, MD simulations and free energy calculations were performed on pyridine amides and 1,2,4-triazolopyridines as 11ß-HSD1 inhibitors to explore structure-activity relationships and structural requirement for the inhibitory activity. 3D-QSAR models, including CoMFA and CoMSIA, were developed from the conformations obtained by docking strategy. The derived pharmacophoric features were further supported by MESP and Mulliken charge analyses using density functional theory. In addition, MD simulations and free energy calculations were employed to determine the detailed binding process and to compare the binding modes of inhibitors with different bioactivities. The binding free energies calculated by MM/PBSA showed a good correlation with the experimental biological activities. Free energy analyses and per-residue energy decomposition indicated the van der Waals interaction would be the major driving force for the interactions between an inhibitor and 11ß-HSD1. These unified results may provide that hydrogen bond interactions with Ser170 and Tyr183 are favorable for enhancing activity. Thr124, Ser170, Tyr177, Tyr183, Val227, and Val231 are the key amino acid residues in the binding pocket. The obtained results are expected to be valuable for the rational design of novel potent 11ß-HSD1 inhibitors.

Dramatic Number Variation of R Genes in Solanaceae Species Accounted for by a Few R Gene Subfamilies.

Most disease resistance genes encode nucleotide-binding-site (NBS) and leucine-rich-repeat (LRR) domains, and the NBS-LRR encoding genes are often referred to as R genes. Using newly developed approach, 478, 485, 1,194, 1,665, 2,042 and 374 R genes were identified from the genomes of tomato Heinz1706, wild tomato LA716, potato DM1-3, pepper Zunla-1 and wild pepper Chiltepin and tobacco TN90, respectively. The majority of R genes from Solanaceae were grouped into 87 subfamilies, including 16 TIR-NBS-LRR (TNL) and 71 non-TNL subfamilies. Each subfamily was annotated manually, including identification of intron/exon structure and intron phase. Interestingly, TNL subfamilies have similar intron phase patterns, while the non-TNL subfamilies have diverse intron phase due to frequent gain of introns. Prevalent presence/absence polymorphic R gene loci were found among Solanaceae species, and an integrated map with 427 R loci was constructed. The pepper genome (2,042 in Chiltepin) has at least four times of R genes as in tomato (478 in Heinz1706). The high number of R genes in pepper genome is due to the amplification of R genes in a few subfamilies, such as the Rpi-blb2 and BS2 subfamilies. The mechanism underlying the variation of R gene number among different plant genomes is discussed.

Molecular modeling studies to characterize N-phenylpyrimidin-2-amine selectivity for CDK2 and CDK4 through 3D-QSAR and molecular dynamics simulations.

CDK2 is a promising target for the development of anti-cancer agents. It is not an easy task to design CDK2-selective inhibitors which do not exhibit activity for other CDK family members, particularly CDK4, due to a high degree of structural homology among CDK family members. In this study, 4-substituted N-phenylpyrimidin-2-amine derivatives as CDK2 inhibitors were examined to understand the selectivity mechanism against CDK4 using a combined approach of 3D-QSAR, molecular docking, MESP, MD simulations, and binding free energy calculations. 3D-QSAR models were developed to propose structural determinants for CDK2 and CDK4 inhibition. High q(2) and r(2) values for CoMFA and CoMSIA models based on both internal and external validations suggested that the generated 3D-QSAR models may exhibit good capability to predict bioactivities of inhibitors against CDK2 or CDK4. Electrostatic potentials on the molecular surface have been discussed in detail for determining the binding affinity of studied inhibitors by combining molecular docking with MESP and Mulliken charge analyses. Binding free energy calculations suggested that the residues Gln85, Asp86, and Lys89 of CDK2 would play a critical role in selective CDK2 inhibition. The electrostatic interactions of an inhibitor with Glu144 and Asn145 of CDK4 may predominately drive CDK4 inhibition. These findings may provide a better structural understanding of the mechanism of CDK2 selective inhibition. The results obtained in the current study may provide valuable guidelines for developing novel potent and selective CDK2 inhibitors.