Identification, molecular evolution, codon bias, and expansion analysis of NLP transcription factor family in foxtail millet (Setaria italica L.) and closely related crops.

The NODULE-INCEPTION-like protein (NLP) family is a plant-specific transcription factor (TF) family involved in nitrate transport and assimilation in plants, which are essential for improving plant nitrogen use efficiency. Currently, the molecular nature and evolutionary trajectory of NLP genes in the C4 model crop foxtail millet are unknown. Therefore, we performed a comprehensive analysis of NLP and molecular evolution in foxtail millet by scanning the genomes of foxtail millet and representative species of the plant kingdom. We identified seven NLP genes in the foxtail millet genome, all of which are individually and separately distributed on different chromosomes. They were not structurally identical to each other and were mainly expressed on root tissues. We unearthed two key genes (Si5G004100.1 and Si6G248300.1) with a variety of excellent characteristics. Regarding its molecular evolution, we found that NLP genes in Gramineae mainly underwent dispersed duplication, but maize NLP genes were mainly generated via WGD events. Other factors such as base mutations and natural selection have combined to promote the evolution of NLP genes. Intriguingly, the family in plants showed a gradual expansion during evolution with more duplications than losses, contrary to most gene families. In conclusion, this study advances the use of NLP genetic resources and the understanding of molecular evolution in cereals.

Proteomic analysis of leaves and roots during drought stress and recovery in Setaria italica L.

Drought is a major environmental factor that limits agricultural crop productivity and threatens food security. Foxtail millet is a model crop with excellent abiotic stress tolerance and is consequently an important subject for obtaining a better understanding of the molecular mechanisms underlying plant responses to drought and recovery. Here the physiological and proteomic responses of foxtail millet (cultivar Yugu1) leaves and roots to drought treatments and recovery were evaluated. Drought-treated foxtail millet exhibited increased relative electrolyte leakage and decreased relative water content and chlorophyll content compared to control and rewatering plants. A global analysis of protein profiles was evaluated for drought-treated and recovery treatment leaves and roots. We also identified differentially abundant proteins in drought and recovery groups, enabling comparisons between leaf and root tissue responses to the conditions. The principal component analysis suggested a clear distinction between leaf and root proteomes for the drought-treated and recovery treatment plants. Gene Ontology enrichment and co-expression analyses indicated that the biological responses of leaves differed from those in roots after drought and drought recovery. These results provide new insights and data resources to investigate the molecular basis of tissue-specific functional responses of foxtail millet during drought and recovery, thereby significantly informing crop breeding.

Integrative genomics analysis of the ever-shrinking pectin methylesterase (PME) gene family in foxtail millet (Setaria italica).

Pectin methylesterase (PME) plays a vital role in the growth and development of plants. Their genes can be classified into two types, with Type-1 having an extra domain, PMEI. PME genes in foxtail millet (Setaria italica L.) have not been identified, and their sequence features and evolution have not been explored. Here, we identified 41 foxtail millet PME genes. Decoding the pro-region, containing the PMEI domain, revealed its more active nature than the DNA encoding PME domain, easier to be lost to produce Type-2 PME genes. We inferred that the active nature of the pro-region could be related to its harbouring more repetitive DNA sequences. Further, we revealed that though whole-genome duplication and tandem duplication contributed to producing new copies of PME genes, phylogenetic analysis provided clear evidence of ever-shrinking gene family size in foxtail millet and the other grasses in the past 100 million years. Phylogenetic analysis also supports the existence of two gene groups, Group I and Group II, with genes in Group II being more conservative. Our research contributes to understanding how DNA sequence structure affects the functional innovation and evolution of PME genes.

Paleopolyploidies and Genomic Fractionation in Major Eudicot Clades.

Eudicots account for ~75% of living angiosperms, containing important food and energy crops. Recently, high-quality genome sequences of several eudicots including Aquilegia coerulea and Nelumbo nucifera have become available, providing an opportunity to investigate the early evolutionary characteristics of eudicots. We performed genomic hierarchical and event-related alignments to infer homology within and between representative species of eudicots. The results provide strong evidence for multiple independent polyploidization events during the early diversification of eudicots, three of which are likely to be allopolyploids: The core eudicot-common hexaploidy (ECH), Nelumbo-specific tetraploidy (NST), and Ranunculales-common tetraploidy (RCT). Using different genomes as references, we constructed genomic alignment to list the orthologous and paralogous genes produced by polyploidization and speciation. This could provide a fundamental framework for studying other eudicot genomes and gene(s) evolution. Further, we revealed significantly divergent evolutionary rates among these species. By performing evolutionary rate correction, we dated RCT to be ~118-134 million years ago (Mya), after Ranunculales diverged with core eudicots at ~123-139 Mya. Moreover, we characterized genomic fractionation resulting from gene loss and retention after polyploidizations. Notably, we revealed a high degree of divergence between subgenomes. In particular, synonymous nucleotide substitutions at synonymous sites (Ks) and phylogenomic analyses implied that A. coerulea might provide the subgenome(s) for the gamma-hexaploid hybridization.

CFVisual: an interactive desktop platform for drawing gene structure and protein architecture.

BACKGROUND: When researchers perform gene family analysis, they often analyze the structural characteristics of the gene, such as the distribution of introns and exons. At the same time, characteristic structural analysis of amino acid sequence is also essential, for example, motif and domain features. Researchers often integrate these analyses into one image to dig out more information, but the tools responsible for this integration are lacking. RESULTS: Here, we developed a tool (CFVisual) for drawing gene structure and protein architecture. CFVisual can draw the phylogenetic tree, gene structure, and protein architecture in one picture, and has rich interactive capabilities, which can meet the work needs of researchers. Furthermore, it also supports arbitrary stitching of the above analysis images. It has become a useful helper in gene family analysis. The CFVisual package was implemented in Python and is freely available from https://github.com/ChenHuilong1223/CFVisual/ . CONCLUSION: CFVisual has been used by some researchers and cited by some articles. In the future, CFVisual will continue to serve as a good helper for researchers in the study of gene structure and protein architecture.

Triplication is the main evolutionary driving force of NLP transcription factor family in Chinese cabbage and related species.

The NODULE-INCEPTION-like protein (NLP) is a plant-specific transcription factor (TF) family that plays an important role in both signal transduction and nitrate assimilation. However, the NLP gene family in Chinese cabbage (Brassica rapa) has yet to be studied. Here we identified 17, 16, and 32 NLP genes in Chinese cabbage, Brassica oleracea, and Brassica napus, respectively. We found that duplication of those NLP genes almost always originated from genome-wide duplication events. Further analysis (using Arabidopsis as a reference) revealed that the NLP family in Chinese cabbage and B. oleracea was characterized by direct expansion caused by whole-genome duplication. By contrast, indirect expansion characterized B. napus, which arose from hybridization and fusion of the two species. In addition, phylogenetic and homology analyses showed that the Brassica NLP gene family has been highly conserved in evolution. Finally, we also identified optimal codons for four studied species. Altogether, through comparative genome analysis methods, we presented compelling evidence that triplication is the main driving force for the NLP TF family's evolution in Chinese cabbage and related Brassica plants, a process evidently highly conserved. This work will help in better understanding the impact of genome-wide duplication on gene families of plants.

Arabidopsis SKU5 Similar 11 and 12 play crucial roles in pollen tube integrity, growth and guidance.

Pollen tube integrity, growth and guidance are crucial factors in plant sexual reproduction. Members of the plant Skewed5 (SKU5) Similar (SKS) family show strong similarity to multicopper oxidases (MCOs), but they lack conserved histidines in MCO active sites. The functions of most SKS family members are unknown. Here, we show that Arabidopsis pollen-expressed SKS11 and SKS12 play important roles in pollen tube integrity, growth and guidance. The sks11sks12 mutant exhibited significantly reduced male fertility. Most of the pollen from sks11sks12 plants burst when germinated, and the pollen tubes grew slowly and exhibited defective growth along the funiculus and micropyle. SKS11-GFP and SKS12-mCherry were detected at the cell wall in pollen tubes. The contents of several cell wall polysaccharides and arabinogalactans were decreased in the pollen tube cell walls of sks11sks12 plants. Staining with a reactive oxygen species (ROS)-sensitive dye and use of the H2 O2 sensor HyPer revealed that the ROS content in the pollen tubes of sks11sks12 plants was remarkably reduced. SKS11444His-Ala , in which the last conserved histidine was mutated, could restore the mutant phenotypes of sks11sks12. Thus, SKS11/12 are required for pollen tube integrity, growth and guidance possibly by regulating the ROS level and cell wall polysaccharide deposition or remodeling in pollen tubes.

Illegitimate Recombination between Duplicated Genes Generated from Recursive Polyploidizations Accelerated the Divergence of the Genus Arachis.

The peanut (Arachis hypogaea L.) is the leading oil and food crop among the legume family. Extensive duplicate gene pairs generated from recursive polyploidizations with high sequence similarity could result from gene conversion, caused by illegitimate DNA recombination. Here, through synteny-based comparisons of two diploid and three tetraploid peanut genomes, we identified the duplicated genes generated from legume common tetraploidy (LCT) and peanut recent allo-tetraploidy (PRT) within genomes. In each peanut genome (or subgenomes), we inferred that 6.8-13.1% of LCT-related and 11.3-16.5% of PRT-related duplicates were affected by gene conversion, in which the LCT-related duplicates were the most affected by partial gene conversion, whereas the PRT-related duplicates were the most affected by whole gene conversion. Notably, we observed the conversion between duplicates as the long-lasting contribution of polyploidizations accelerated the divergence of different Arachis genomes. Moreover, we found that the converted duplicates are unevenly distributed across the chromosomes and are more often near the ends of the chromosomes in each genome. We also confirmed that well-preserved homoeologous chromosome regions may facilitate duplicates' conversion. In addition, we found that these biological functions contain a higher number of preferentially converted genes, such as catalytic activity-related genes. We identified specific domains that are involved in converted genes, implying that conversions are associated with important traits of peanut growth and development.

Identification, Molecular Characteristics, and Evolution of GRF Gene Family in Foxtail Millet (Setaria italica L.).

Growth-regulating factor (GRF) is a multigene family that plays a vital role in the growth and development of plants. In the past, the GRF family of many plants has been studied. However, there is not a report about identification and evolution of GRF in foxtail millet (Setaria italia). Here, we identified 10 GRF genes in foxtail millet. Seven (70.00%) were regulated by Sit-miR396, and there were 19 optimal codons in GRFs of foxtail millet. Additionally, we found that WGD or segmental duplication have affected GRFs in foxtail millet between 15.07 and 45.97 million years ago. Regarding the GRF gene family of land plants, we identified a total of 157 GRF genes in 15 representative land plants. We found that GRF gene family originated from Group E, and the GRF gene family in monocots was gradually shrinking. Also, more loss resulted from the small number of GRF genes in lower plants. Exploring the evolution of GRF and functional analysis in the foxtail millet help us to understand GRF better and make a further study about the mechanism of GRF. These results provide a basis for the genetic improvement of foxtail millet and indicate an improvement of the yield.

Deciphering the high-quality genome sequence of coriander that causes controversial feelings.

Coriander (Coriandrum sativum L. 2n = 2x = 22), a plant from the Apiaceae family, also called cilantro or Chinese parsley, is a globally important crop used as vegetable, spice, fragrance and traditional medicine. Here, we report a high-quality assembly and analysis of its genome sequence, anchored to 11 chromosomes, with total length of 2118.68 Mb and N50 scaffold length of 160.99 Mb. We found that two whole-genome duplication events, respectively, dated to ~45-52 and ~54-61 million years ago, were shared by the Apiaceae family after their split from lettuce. Unbalanced gene loss and expression are observed between duplicated copies produced by these two events. Gene retention, expression, metabolomics and comparative genomic analyses of terpene synthase (TPS) gene family, involved in terpenoid biosynthesis pathway contributing to coriander's special flavour, revealed that tandem duplication contributed to coriander TPS gene family expansion, especially compared to their carrot counterparts. Notably, a TPS gene highly expressed in all 4 tissues and 3 development stages studied is likely a major-effect gene encoding linalool synthase and myrcene synthase. The present genome sequencing, transcriptome, metabolome and comparative genomic efforts provide valuable insights into the genome evolution and spice trait biology of Apiaceae and other related plants, and facilitated further research into important gene functions and crop improvement.

Polyploidy Index and Its Implications for the Evolution of Polyploids.

Polyploidy has contributed to the divergence and domestication of plants; however, estimation of the relative roles that different types of polyploidy have played during evolution has been difficult. Unbalanced and balanced gene removal was previously related to allopolyploidies and autopolyploidies, respectively. Here, to infer the types of polyploidies and evaluate their evolutionary effects, we devised a statistic, the Polyploidy-index or P-index, to characterize the degree of divergence between subgenomes of a polyploidy, to find whether there has been a balanced or unbalanced gene removal from the homoeologous regions. Based on a P-index threshold of 0.3 that distinguishes between known or previously inferred allo- or autopolyploidies, we found that 87.5% of 24 angiosperm paleo-polyploidies were likely produced by allopolyploidizations, responsible for establishment of major tribes such as Poaceae and Fabaceae, and large groups such as monocots and eudicots. These findings suggest that >99.7% of plant genomes likely derived directly from allopolyploidies, with autopolyploidies responsible for the establishment of only a few small genera, including Glycine, Malus, and Populus, each containing tens of species. Overall, these findings show that polyploids with high divergence between subgenomes (presumably allopolyploids) established the major plant groups, possibly through secondary contact between previously isolated populations and hybrid vigor associated with their re-joining.

Whole RNA-sequencing and gene expression analysis of Trichoderma harzianum Tr-92 under chlamydospore-producing condition.

BACKGROUND: Trichoderma is one of the most important biocontrol fungi, which could produce mycelia, conidiospores, and chlamydospores three types of propagules under different conditions. Chlamydospores are produced in harsh conditions in various fungi, and may be more resistant to adverse conditions. However, the knowledge associated with the mechanism of chlamydospore formation remained unclear in Trichoderma. OBJECTIVES: This study is aimed to explore the essential genes and regulatory pathways associated with chlamydospore formation in Trichoderma. METHODS: The culture condition, survival rate, and biocontrol effects of chlamydospores and conidiospores from Trichoderma.harzianum Tr-92 were determined. Furthermore, the whole transcriptome profiles of T. harzianum Tr-92 under chlamydospore-producing and chlamydospore-nonproducing conditions were performed. RESULTS: T. harzianum Tr-92 produced chlamydospores under particular conditions, and chlamydospore-based formulation of T. harzianum Tr-92 exhibited higher biocontrol ability against Botrytis cinerea in cucumber than conidoiospore-based formulation. In the transcriptome analysis, a total of 2,029 differentially expressed genes (DEGs) were identified in T. harzianum Tr-92 under chlamydospore-producing condition, compared to that under chlamydospore-nonproducing condition. GO classification indicated that the DEGs were significantly enriched in 284 terms among biological process, cellular components and molecular function categories. A total of 19 pathways were observed with DEGs by KEGG analysis. Furthermore, fifteen DEGs were verified by quantitative real-time PCR, and the expression profiles were consistent with the transcriptome data. CONCLUSION: The results would provide a basis on the molecular mechanisms underlying Trichoderma sporulation, which would assist the development and application of fungal biocontrol agents.

Reconstruction of evolutionary trajectories of chromosomes unraveled independent genomic repatterning between Triticeae and Brachypodium.

BACKGROUND: After polyploidization, a genome may experience large-scale genome-repatterning, featuring wide-spread DNA rearrangement and loss, and often chromosome number reduction. Grasses share a common tetraploidization, after which the originally doubled chromosome numbers reduced to different chromosome numbers among them. A telomere-centric reduction model was proposed previously to explain chromosome number reduction. With Brachpodium as an intermediate linking different major lineages of grasses and a model plant of the Pooideae plants, we wonder whether it mediated the evolution from ancestral grass karyotype to Triticeae karyotype. RESULTS: By inferring the homology among Triticeae, rice, and Brachpodium chromosomes, we reconstructed the evolutionary trajectories of the Triticeae chromosomes. By performing comparative genomics analysis with rice as a reference, we reconstructed the evolutionary trajectories of Pooideae plants, including Ae. Tauschii (2n = 14, DD), barley (2n = 14), Triticum turgidum (2n = 4x = 28, AABB), and Brachypodium (2n = 10). Their extant Pooidea and Brachypodium chromosomes were independently produced after sequential nested chromosome fusions in the last tens of millions of years, respectively, after their split from rice. More frequently than would be expected by chance, in Brachypodium, the 'invading' and 'invaded' chromosomes are homoeologs, originating from duplication of a common ancestral chromosome, that is, with more extensive DNA-level correspondence to one another than random chromosomes, nested chromosome fusion events between homoeologs account for three of seven cases in Brachypodium (P-value≈0.00078). However, this phenomenon was not observed during the formation of other Pooideae chromosomes. CONCLUSIONS: Notably, we found that the Brachypodium chromosomes formed through exclusively distinctive trajectories from those of Pooideae plants, and were well explained by the telomere-centric model. Our work will contribute to understanding the structural and functional innovation of chromosomes in different Pooideae lineages and beyond.

Involvement of jasmonic acid, ethylene and salicylic acid signaling pathways behind the systemic resistance induced by Trichoderma longibrachiatum H9 in cucumber.

BACKGROUND: Trichoderma spp. are effective biocontrol agents for many plant pathogens, thus the mechanism of Trichoderma-induced plant resistance is not fully understood. In this study, a novel Trichoderma strain was identified, which could promote plant growth and reduce the disease index of gray mold caused by Botrytis cinerea in cucumber. To assess the impact of Trichoderma inoculation on the plant response, a multi-omics approach was performed in the Trichoderma-inoculated cucumber plants through the analyses of the plant transcriptome, proteome, and phytohormone content. RESULTS: A novel Trichoderma strain was identified by morphological and molecular analysis, here named T. longibrachiatum H9. Inoculation of T. longibrachiatum H9 to cucumber roots promoted plant growth in terms of root length, plant height, and fresh weight. Root colonization of T. longibrachiatum H9 in the outer layer of epidermis significantly inhibited the foliar pathogen B. cinerea infection in cucumber. The plant transcriptome and proteome analyses indicated that a large number of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were identified in cucumber plants 96 h post T. longibrachiatum H9 inoculation. Up-regulated DEGs and DEPs were mainly associated with defense/stress processes, secondary metabolism, and phytohormone synthesis and signaling, including jasmonic acid (JA), ethylene (ET) and salicylic acid (SA), in the T. longibrachiatum H9-inoculated cucumber plants in comparison to untreated plants. Moreover, the JA and SA contents significantly increased in cucumber plants with T. longibrachiatum H9 inoculation. CONCLUSIONS: Application of T. longibrachiatum H9 to the roots of cucumber plants effectively promoted plant growth and significantly reduced the disease index of gray mold caused by B. cinerea. The analyses of the plant transcriptome, proteome and phytohormone content demonstrated that T. longibrachiatum H9 mediated plant systemic resistance to B. cinerea challenge through the activation of signaling pathways associated with the phytohormones JA/ET and SA in cucumber.

Recursive Paleohexaploidization Shaped the Durian Genome.

The durian (Durio zibethinus) genome has recently become available, and analysis of this genome reveals two paleopolyploidization events previously inferred as shared with cotton (Gossypium spp.). Here, we reanalyzed the durian genome in comparison with other well-characterized genomes. We found that durian and cotton were actually affected by different polyploidization events: hexaploidization in durian ∼19-21 million years ago (mya) and decaploidization in cotton ∼13-14 mya. Previous interpretations of shared polyploidization events may have resulted from the elevated evolutionary rates in cotton genes due to the decaploidization and insufficient consideration of the complexity of plant genomes. The decaploidization elevated evolutionary rates of cotton genes by ∼64% compared to durian and explained a previous ∼4-fold over dating of the event. In contrast, the hexaploidization in durian did not prominently elevate gene evolutionary rates, likely due to its long generation time. Moreover, divergent evolutionary rates probably explain 98.4% of reconstructed phylogenetic trees of homologous genes being incongruent with expected topology. The findings provide further insight into the roles played by polypoidization in the evolution of genomes and genes, and they suggest revisiting existing reconstructed phylogenetic trees.

Genomic, expressional, protein-protein interactional analysis of Trihelix transcription factor genes in Setaria italia and inference of their evolutionary trajectory.

BACKGROUND: Trihelix transcription factors (TTF) play important roles in plant growth and response to adversity stress. Until now, genome-wide identification and analysis of this gene family in foxtail millet has not been available. Here, we identified TTF genes in the foxtail millet and its grass relatives, and characterized their functional domains. RESULTS: As to sequence divergence, TTF genes were previously divided into five subfamilies, I-V. We found that Trihelix family members in foxtail millet and other grasses mostly preserved their ancestral chromosomal locations during millions of years' evolution. Six amino acid sites of the SIP1 subfamily possibly were likely subjected to significant positive selection. Highest expression level was observed in the spica, with the SIP1 subfamily having highest expression level. As to the origination and expansion of the gene family, notably we showed that a subgroup of subfamily IV was the oldest, and therefore was separated to define a new subfamily O. Overtime, starting from the subfamily O, certain genes evolved to form subfamilies III and I, and later from subfamily I to develop subfamilies II and V. The oldest gene, Si1g016284, has the most structural changes, and a high expression in different tissues. What's more interesting is that it may have bridge the interaction with different proteins. CONCLUSIONS: By performing phylogenetic analysis using non-plant species, notably we showed that a subgroup of subfamily IV was the oldest, and therefore was separated to define a new subfamily O. Starting from the subfamily O, certain genes evolved to form other subfamilies. Our work will contribute to understanding the structural and functional innovation of Trihelix transcription factor, and the evolutionary trajectory.

Two Likely Auto-Tetraploidization Events Shaped Kiwifruit Genome and Contributed to Establishment of the Actinidiaceae Family.

The genome of kiwifruit (Actinidia chinensis) was sequenced previously, the first in the Actinidiaceae family. It was shown to have been affected by polyploidization events, the nature of which has been elusive. Here, we performed a reanalysis of the genome and found clear evidence of 2 tetraploidization events, with one occurring ∼50-57 million years ago (Mya) and the other ∼18-20 Mya. Two subgenomes produced by each event have been under balanced fractionation. Moreover, genes were revealed to express in a balanced way between duplicated copies of chromosomes. Besides, lowered evolutionary rates of kiwifruit genes were observed. These findings could be explained by the likely auto-tetraploidization nature of the polyploidization events. Besides, we found that polyploidy contributed to the expansion of key functional genes, e.g., vitamin C biosynthesis genes. The present work also provided an important comparative genomics resource in the Actinidiaceae and related families.

Comprehensive analyses of the BES1 gene family in Brassica napus and examination of their evolutionary pattern in representative species.

BACKGROUND: The BES1 gene family, an important class of plant-specific transcription factors, play key roles in the BR signal pathway in plants, regulating various development processes. Until now, there has been no comprehensive analysis of the BES1 gene family in Brassica napus, and a cross-genome exploration of their origin, copy number changes, and functional innovation in plants was also not available. RESULTS: We identified 28 BES1 genes in B. napus from its two subgenomes (AA and CC). We found that 71.43% of them were duplicated in the tetraploidization, and their gene expression showed a prominent subgenome bias in the roots. Additionally, we identified 104 BES1 genes in another 18 representative angiosperms and performed a comparative analysis with B. napus, including evolutionary trajectory, gene duplication, positive selection, and expression pattern. Exploiting the available genome datasets, we performed a large-scale analysis across plants and algae suggested that the BES1 gene family could have originated from group F, expanding to form other groups (A to E) by duplicating or alternatively deleting some domains. We detected an additional domain containing M4 to M8 in exclusively groups F1 and F2. We found evidence that whole-genome duplication (WGD) contributed the most to the expansion of this gene family among examined dicots, while dispersed duplication contributed the most to expansion in certain monocots. Moreover, we inferred that positive selection might have occurred on major phylogenetic nodes during the evolution of plants. CONCLUSIONS: Grossly, a cross-genome comparative analysis of the BES1 genes in B. napus and other species sheds light on understanding its copy number expansion, natural selection, and functional innovation.

Pollen-Expressed Leucine-Rich Repeat Extensins Are Essential for Pollen Germination and Growth.

During pollen tube growth, the walls of the tube provide the mechanical strength resisting turgor pressure to protect two sperm cells. Cell wall proteins may play an important role in this process. Pollen tube cell wall proteins known as leucine-rich repeat extensins (LRXs) harbor a leucine-rich repeat domain and an extensin domain. In this study, the functions of four pollen-expressed LRXs, LRX8, LRX9, LRX10, and LRX11 (LRX8-11), were characterized in Arabidopsis (Arabidopsis thaliana). LRX8-11 displayed a consistent expression pattern in mature pollen grains and pollen tubes. In a phenotypic analysis of four single mutants, six double mutants, four triple mutants, and a quadruple mutant, the triple and quadruple mutant plants displayed markedly reduced seed set and decreased male transmission efficiency accompanied by compromised pollen germination and pollen tube growth. GFP-fused LRX8, LRX10, and LRX11 were found to be localized to pollen tube cell walls. An immunohistochemical analysis of pollen tube cell wall polysaccharides showed an increase in the amount of rhamnogalacturonan I in the subapical walls of pollen tubes of the lrx9 lrx10 lrx11 and lrx8 lrx9 lrx11 mutants and a decrease in the content of fucosylated xyloglucans in lrx8 lrx9 lrx11 compared with wild-type plants. Moreover, the callose content in the apical walls of pollen tubes increased in the lrx8 lrx9 lrx11 mutant. In conclusion, we propose that LRX8-11 function synergistically to maintain pollen tube cell wall integrity; thus, they play critical roles in pollen germination and pollen tube growth.

An Overlooked Paleotetraploidization in Cucurbitaceae.

Cucurbitaceae plants are of considerable biological and economic importance, and genomes of cucumber, watermelon, and melon have been sequenced. However, a comparative genomics exploration of their genome structures and evolution has not been available. Here, we aimed at performing a hierarchical inference of genomic homology resulted from recursive paleopolyploidizations. Unexpectedly, we found that, shortly after a core-eudicot-common hexaploidy, a cucurbit-common tetraploidization (CCT) occurred, overlooked by previous reports. Moreover, we characterized gene loss (and retention) after these respective events, which were significantly unbalanced between inferred subgenomes, and between plants after their split. The inference of a dominant subgenome and a sensitive one suggested an allotetraploid nature of the CCT. Besides, we found divergent evolutionary rates among cucurbits, and after doing rate correction, we dated the CCT to be 90-102 Ma, likely common to all Cucurbitaceae plants, showing its important role in the establishment of the plant family.