Genomic and transcriptomic analyses provide insights into valuable fatty acid biosynthesis and environmental adaptation of yellowhorn.

Yellowhorn (Xanthoceras sorbifolium) is an oil-bearing tree species growing naturally in poor soil. The kernel of yellowhorn contains valuable fatty acids like nervonic acid. However, the genetic basis underlying the biosynthesis of valued fatty acids and adaptation to harsh environments is mainly unexplored in yellowhorn. Here, we presented a haplotype-resolved chromosome-scale genome assembly of yellowhorn with the size of 490.44 Mb containing scaffold N50 of 34.27 Mb. Comparative genomics, in combination with transcriptome profiling analyses, showed that expansion of gene families like long-chain acyl-CoA synthetase and ankyrins contribute to yellowhorn fatty acid biosynthesis and defense against abiotic stresses, respectively. By integrating genomic and transcriptomic data of yellowhorn, we found that the transcription of 3-ketoacyl-CoA synthase gene XS04G00959 was consistent with the accumulation of nervonic and erucic acid biosynthesis, suggesting its critical regulatory roles in their biosynthesis. Collectively, these results enhance our understanding of the genetic basis underlying the biosynthesis of valuable fatty acids and adaptation to harsh environments in yellowhorn and provide foundations for its genetic improvement.

Regulation of WOX11 Expression Represents the Difference Between Direct and Indirect Shoot Regeneration.

Somatic cells of higher plants possess the remarkable ability to regenerate new individuals via reestablishing apical meristems. Reconstitution of shoot meristem is the vital process and is required for application of plant biotechnology. Under in vitro culture condition, shoot meristem can be formed directly or indirectly, depending on the absence or presence of callus as the intermediate status. However, the difference of regulatory mechanisms between the two regeneration types remains unknown. In this study, we established a bi-directional system in which shoots regenerated directly from lateral root primordia (LRP) and indirectly from hypocotyl-derived callus simultaneously. The results based on this system revealed that regulation of WOX11 expression represents the difference between the two regeneration types in two aspects. Firstly, number of founder cells expressing WOX11 is tightly associated with regeneration types. Relatively more founder cells gave rise to callus and produce larger meristem, whereas less founder cells produce LRP that regenerate smaller meristem. Secondly, non-CG DNA methylation specifically regulated WOX11 transcription in LRP and promoted direct shoot regeneration, but had no influence on indirect regeneration. The results provide new insights for understanding the regulatory mechanisms of cell fate transition during de novo organogenesis.

ARF4 regulates shoot regeneration through coordination with ARF5 and IAA12.

KEY MESSAGE: ARF4-regulated shoot regeneration through competing with ARF5 for the interaction with IAA12. Plant possess the ability to regenerate shoot meristem and subsequent the whole individual. This process is the foundation for in vitro propagation and genetic engineering and provides a system for studying fundamental biological questions, such as hormonal signaling. Auxin response factor (ARF) family transcription factors are critical components of auxin signaling pathway that regulate the transcription of target genes. To date, the mechanisms underlying the functions of class-B ARFs which act as transcription repressors remains unclear. In this study, we found that ARF4, the transcriptional repressor, was involved in regulating shoot regeneration. ARF4 interacted with auxin/Indole-3-Acetic-Acid12 (IAA12). The expression signals of ARF4 displayed a dynamic pattern similar with those of ARF5 and IAA12 during shoot meristem formation. Enhanced expression of IAA12 compromised the shoot regeneration capacity. Induced expression of ARF4 complemented the regeneration phenotype of IAA12-overexpression but did not rescued the defects in the arf5 mutant, mp-S319. Further analysis revealed that ARF4 competed with ARF5 for the interaction with IAA12. The results indicate that ARF4-regulated shoot regeneration through cooperating with ARF5 and IAA12. Our findings provided new information for deciphering the function of class-B ARFs.

The genome assembly and annotation of yellowhorn (Xanthoceras sorbifolium Bunge).

BACKGROUND: Yellowhorn (Xanthoceras sorbifolium Bunge), a deciduous shrub or small tree native to north China, is of great economic value. Seeds of yellowhorn are rich in oil containing unsaturated long-chain fatty acids that have been used for producing edible oil and nervonic acid capsules. However, the lack of a high-quality genome sequence hampers the understanding of its evolution and gene functions. FINDINGS: In this study, a whole genome of yellowhorn was sequenced and assembled by integration of Illumina sequencing, Pacific Biosciences single-molecule real-time sequencing, 10X Genomics linked reads, Bionano optical maps, and Hi-C. The yellowhorn genome assembly was 439.97 Mb, which comprised 15 pseudo-chromosomes covering 95.42% (419.84 Mb) of the assembled genome. The repetitive fractions accounted for 56.39% of the yellowhorn genome. The genome contained 21,059 protein-coding genes. Of them, 18,503 (87.86%) genes were found to be functionally annotated with ≥1 "annotation" term by searching against other databases. Transcriptomic analysis showed that 341, 135, 125, 113, and 100 genes were specifically expressed in hermaphrodite flower, staminate flower, young fruit, leaf, and shoot, respectively. Phylogenetic analysis suggested that yellowhorn and Dimocarpus longan diverged from their most recent common ancestor ∼46 million years ago. CONCLUSIONS: The availability and subsequent annotation of the yellowhorn genome, as well as the identification of tissue-specific functional genes, provides a valuable reference for plant comparative genomics, evolutionary studies, and molecular design breeding.

iPSCs: A Comparison between Animals and Plants.

Pluripotent stem cells (PSCs) are self-renewable cells with the potential to differentiate into all the cell types within an organism. PSCs exist transiently in early-stage mammalian embryos during ontogeny and are maintained in apical meristems of higher plants throughout postembryonic development. Through proper in vitro culture, somatic cells of both mammals and plants can be reprogrammed to generate induced PSCs (iPSCs). Recent studies have deciphered mechanisms underlying pluripotency gene activation and cell fate transition during plant iPSC generation. Here, we compare these mechanisms with those of their animal counterparts in the hope that this may trigger mutual learning of researchers from both fields, leading to advances and independent breakthroughs in this important area.

Plant stem cells and de novo organogenesis.

Contents Summary 1334 I. Introduction 1334 II. Regeneration-initial cell: the origin of regeneration 1335 III. Acquiring regeneration competency: the essential intermediate step for hormone-induced regeneration 1335 IV. Hormonal induction of stem cell regulators: the program for de novo establishment of apical meristems 1337 V. Conclusions and perspectives 1337 Acknowledgements 1338 Author contributions 1338 References 1338 SUMMARY: High cellular plasticity confers remarkable regeneration capacity to plants. Based on the activity of stem cells and their regulators, higher plants are capable of regenerating new individuals. De novo organogenesis exemplifies the regeneration of the whole plant body and is exploited widely in agriculture and biotechnology. In this Tansley insight article, we summarize recent advances that facilitate our understanding of the molecular mechanisms underlying de novo organogenesis. According to our current knowledge, this process can be divided into three steps, including activation of regeneration-initial cells, acquisition of competency and de novo establishment of apical meristems. The functions of stem cells and their regulators are critical to de novo organogenesis, whereas auxin and cytokinin act as triggers and linkers between different steps.

Type-B ARRs Control Carpel Regeneration Through Mediating AGAMOUS Expression in Arabidopsis.

Plants are known for their capacity to regenerate organs, such as shoot, root and floral organs. Recently, a number of studies contributed to understanding the mechanisms of shoot and root regeneration. However, the mechanisms underlying floral organ regeneration are largely unknown. In this study, we established a carpel regeneration system in which two types of carpels were induced by exogenous cytokinin. For type I, all the floral organs in the regenerated inflorescence were transformed into carpels. For type II, carpels were generated directly from callus. The transcript level of AGAMOUS (AG), the carpel identity gene, was up-regulated during carpel induction. The expression signals of AG were detected in the initiating carpel primordia and regenerating carpels, and co-localized with those of two Type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), ARR1 and ARR10. Repression of either AG or type-B ARRs reduced carpel regeneration. Binding analyses showed that ARR1 and ARR10 directly bound to transcriptional regulatory regions of AG and positively regulated its expression. In addition, the expression of type-B ARRs overlapped with that of AG in the floral primordia in planta. Defects in type-B ARRs reduced the number of carpels. The results indicate that type-B ARRs control carpel regeneration through activating AG expression. Our results provide new information for understanding the mechanism of carpel formation.

Genome-wide analysis of SSR and ILP markers in trees: diversity profiling, alternate distribution, and applications in duplication.

Molecular markers are efficient tools for breeding and genetic studies. However, despite their ecological and economic importance, their development and application have long been hampered. In this study, we identified 524,170 simple sequence repeat (SSR), 267,636 intron length polymorphism (ILP), and 11,872 potential intron polymorphism (PIP) markers from 16 tree species based on recently available genome sequences. Larger motifs, including hexamers and heptamers, accounted for most of the seven different types of SSR loci. Within these loci, A/T bases comprised a significantly larger proportion of sequence than G/C. SSR and ILP markers exhibited an alternative distribution pattern. Most SSRs were monomorphic markers, and the proportions of polymorphic markers were positively correlated with genome size. By verifying with all 16 tree species, 54 SSR, 418 ILP, and four PIP universal markers were obtained, and their efficiency was examined by PCR. A combination of five SSR and six ILP markers were used for the phylogenetic analysis of 30 willow samples, revealing a positive correlation between genetic diversity and geographic distance. We also found that SSRs can be used as tools for duplication analysis. Our findings provide important foundations for the development of breeding and genetic studies in tree species.

Type-B ARABIDOPSIS RESPONSE REGULATORs Specify the Shoot Stem Cell Niche by Dual Regulation of WUSCHEL.

Plants are known for their capacity to regenerate the whole body through de novo formation of apical meristems from a mass of proliferating cells named callus. Exogenous cytokinin and auxin determine cell fate for the establishment of the stem cell niche, which is the vital step of shoot regeneration, but the underlying mechanisms remain unclear. Here, we show that type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), critical components of cytokinin signaling, activate the transcription of WUSCHEL (WUS), which encodes a key regulator for maintaining stem cells. In parallel, type-B ARRs inhibit auxin accumulation by repressing the expression of YUCCAs, which encode a key enzyme for auxin biosynthesis, indirectly promoting WUS induction. Both pathways are essential for de novo regeneration of the shoot stem cell niche. In addition, the dual regulation of type-B ARRs on WUS transcription is required for the maintenance of the shoot apical meristem in planta. Thus, our results reveal a long-standing missing link between cytokinin signaling and WUS regulator, and the findings provide critical information for understanding cell fate specification.

Expression analysis of genes encoding mitogen-activated protein kinases in maize provides a key link between abiotic stress signaling and plant reproduction.

Mitogen-activated protein kinases (MAPKs) play important roles in stress responses and development in plants. Maize (Zea mays), an important cereal crop, is a model plant species for molecular studies. In the last decade, several MAPKs have been identified in maize; however, their functions have not been studied extensively. Genome-wide identification and expression analysis of maize MAPK genes could provide valuable information for understanding their functions. In this study, 20 non-redundant maize MAPK genes (ZmMPKs) were identified via a genome-wide survey. Phylogenetic analysis of MAPKs from maize, rice (Oryza sativa), Arabidopsis (Arabidopsis thaliana), poplar (Populus trichocarpa), and tomato (Solanum lycopersicum) classified them into four major classes. ZmMPKs in the same class had similar domains, motifs, and genomic structures. Gene duplication investigations suggested that segmental duplications made a large contribution to the expansion of ZmMPKs. A number of cis-acting elements related to plant development and response to stress and hormones were identified in the promoter regions of ZmMPKs. Furthermore, transcript profile analysis in eight tissues and organs at various developmental stages demonstrated that most ZmMPKs were preferentially expressed in reproductive tissues and organs. The transcript abundance of most ZmMPKs changed significantly under salt, drought, cold, or abscisic acid (ABA) treatments, implying that they might participate in abiotic stress and ABA signaling. These expression analyses indicated that ZmMPKs might serve as linkers between abiotic stress signaling and plant reproduction. Our data will deepen our understanding of the complexity of the maize MAPK gene family and provide new clues to investigate their functions.

High-throughput sequencing of small RNAs from pollen and silk and characterization of miRNAs as candidate factors involved in pollen-silk interactions in maize.

In angiosperms, successful pollen-pistil interactions are the prerequisite and guarantee of subsequent fertilization and seed production. Recent profile analyses have helped elucidate molecular mechanisms underlying these processes at both transcriptomic and proteomic levels, but the involvement of miRNAs in pollen-pistil interactions is still speculative. In this study, we sequenced four small RNA libraries derived from mature pollen, in vitro germinated pollen, mature silks, and pollinated silks of maize (Zea mays L.). We identified 161 known miRNAs belonging to 27 families and 82 novel miRNAs. Of these, 40 conserved and 16 novel miRNAs showed different expression levels between mature and germinated pollen, and 30 conserved and eight novel miRNAs were differentially expressed between mature and pollinated silks. As candidates for factors associated with pollen-silk (pistil) interactions, expression patterns of the two sets of differentially expressed miRNAs were confirmed by stem-loop real-time RT-PCR. Transcript levels of 22 predicted target genes were also validated using real-time RT-PCR; most of these exhibited expression patterns contrasting with those of their corresponding miRNAs. In addition, GO analysis of target genes of differentially expressed miRNAs revealed that functional categories related to auxin signal transduction and gene expression regulation were overrepresented. These results suggest that miRNA-mediated auxin signal transduction and transcriptional regulation have roles in pollen-silk interactions. The results of our study provide novel information for understanding miRNA regulatory roles in pollen-pistil interactions.

Identification of genes specifically or preferentially expressed in maize silk reveals similarity and diversity in transcript abundance of different dry stigmas.

BACKGROUND: In plants, pollination is a critical step in reproduction. During pollination, constant communication between male pollen and the female stigma is required for pollen adhesion, germination, and tube growth. The detailed mechanisms of stigma-mediated reproductive processes, however, remain largely unknown. Maize (Zea mays L.), one of the world's most important crops, has been extensively used as a model species to study molecular mechanisms of pollen and stigma interaction. A comprehensive analysis of maize silk transcriptome may provide valuable information for investigating stigma functionality. A comparative analysis of expression profiles between maize silk and dry stigmas of other species might reveal conserved and diverse mechanisms that underlie stigma-mediated reproductive processes in various plant species. RESULTS: Transcript abundance profiles of mature silk, mature pollen, mature ovary, and seedling were investigated using RNA-seq. By comparing the transcriptomes of these tissues, we identified 1,427 genes specifically or preferentially expressed in maize silk. Bioinformatic analyses of these genes revealed many genes with known functions in plant reproduction as well as novel candidate genes that encode amino acid transporters, peptide and oligopeptide transporters, and cysteine-rich receptor-like kinases. In addition, comparison of gene sets specifically or preferentially expressed in stigmas of maize, rice (Oryza sativa L.), and Arabidopsis (Arabidopsis thaliana [L.] Heynh.) identified a number of homologous genes involved either in pollen adhesion, hydration, and germination or in initial growth and penetration of pollen tubes into the stigma surface. The comparison also indicated that maize shares a more similar profile and larger number of conserved genes with rice than with Arabidopsis, and that amino acid and lipid transport-related genes are distinctively overrepresented in maize. CONCLUSIONS: Many of the novel genes uncovered in this study are potentially involved in stigma-mediated reproductive processes, including genes encoding amino acid transporters, peptide and oligopeptide transporters, and cysteine-rich receptor-like kinases. The data also suggest that dry stigmas share similar mechanisms at early stages of pollen-stigma interaction. Compared with Arabidopsis, maize and rice appear to have more conserved functional mechanisms. Genes involved in amino acid and lipid transport may be responsible for mechanisms in the reproductive process that are unique to maize silk.

Comparative proteomic analysis reveals similar and distinct features of proteins in dry and wet stigmas.

Angiosperm stigma supports compatible pollen germination and tube growth, resulting in fertilization and seed production. Stigmas are mainly divided into two types, dry and wet, according to the absence or presence of exudates on their surfaces. Here, we used 2DE and MS to identify proteins specifically and preferentially expressed in the stigmas of maize (Zea Mays, dry stigma) and tobacco (Nicotiana tabacum, wet stigma), as well as proteins rinsed from the surface of the tobacco stigma. We found that the specifically and preferentially expressed proteins in maize and tobacco stigmas share similar distributions in functional categories. However, these proteins showed important difference between dry and wet stigmas in a few aspects, such as protein homology in "signal transduction" and "lipid metabolism," relative expression levels of proteins containing signal peptides and proteins in "defense and stress response." These different features might be related to the specific structures and functions of dry and wet stigmas. The possible roles of some stigma-expressed proteins were discussed. Our results provide important information on functions of proteins in dry and wet stigmas and reveal aspects of conservation and divergence between dry and wet stigmas at the proteomic level.