Whole-genome sequencing of diverse wheat accessions uncovers genetic changes during modern breeding in China and the United States.

Breeding has dramatically changed the plant architecture of wheat (Triticum aestivum), resulting in the development of high-yielding varieties adapted to modern farming systems. However, how wheat breeding shaped the genomic architecture of this crop remains poorly understood. Here, we performed a comprehensive comparative analysis of a whole-genome resequencing panel of 355 common wheat accessions (representing diverse landraces and modern cultivars from China and the United States) at the phenotypic and genomic levels. The genetic diversity of modern wheat cultivars was clearly reduced compared to landraces. Consistent with these genetic changes, most phenotypes of cultivars from China and the United States were significantly altered. Of the 21 agronomic traits investigated, 8 showed convergent changes between the 2 countries. Moreover, of the 207 loci associated with these 21 traits, more than half overlapped with genomic regions that showed evidence of selection. The distribution of selected loci between the Chinese and American cultivars suggests that breeding for increased productivity in these 2 regions was accomplished by pyramiding both shared and region-specific variants. This work provides a framework to understand the genetic architecture of the adaptation of wheat to diverse agricultural production environments, as well as guidelines for optimizing breeding strategies to design better wheat varieties.

A Quantitative Computational Framework for Allopolyploid Single-Cell Data Integration and Core Gene Ranking in Development.

Polyploidization drives regulatory and phenotypic innovation. How the merger of different genomes contributes to polyploid development is a fundamental issue in evolutionary developmental biology and breeding research. Clarifying this issue is challenging because of genome complexity and the difficulty in tracking stochastic subgenome divergence during development. Recent single-cell sequencing techniques enabled probing subgenome-divergent regulation in the context of cellular differentiation. However, analyzing single-cell data suffers from high error rates due to high dimensionality, noise, and sparsity, and the errors stack up in polyploid analysis due to the increased dimensionality of comparisons between subgenomes of each cell, hindering deeper mechanistic understandings. In this study, we develop a quantitative computational framework, called "pseudo-genome divergence quantification" (pgDQ), for quantifying and tracking subgenome divergence directly at the cellular level. Further comparing with cellular differentiation trajectories derived from single-cell RNA sequencing data allows for an examination of the relationship between subgenome divergence and the progression of development. pgDQ produces robust results and is insensitive to data dropout and noise, avoiding high error rates due to multiple comparisons of genes, cells, and subgenomes. A statistical diagnostic approach is proposed to identify genes that are central to subgenome divergence during development, which facilitates the integration of different data modalities, enabling the identification of factors and pathways that mediate subgenome-divergent activity during development. Case studies have demonstrated that applying pgDQ to single-cell and bulk tissue transcriptomic data promotes a systematic and deeper understanding of how dynamic subgenome divergence contributes to developmental trajectories in polyploid evolution.

Natural variant of Rht27, a dwarfing gene, enhances yield potential in wheat.

KEY MESSAGE: Discovery of Rht27, a dwarf gene in wheat, showed potential in enhancing grain yield by reducing plant height. Plant height plays a crucial role in crop architecture and grain yield, and semi-dwarf Reduced Height (Rht) alleles contribute to lodging resistance and were important in "Green Revolution." However, the use of these alleles is associated with some negative side effects in some environments, such as reduced coleoptile length, low nitrogen use efficiency, and reduced yield. Therefore, novel dwarf gene resources are needed to pave an alternative route to overcome these side effects. In this study, a super-dwarf mutant rht27 was obtained by the mutagenesis of G1812 (Triticum urartu, the progenitor of the A sub-genome of common wheat). Genetic analysis revealed that the dwarf phenotype was regulated by a single recessive genetic factor. The candidate region for Rht27 was narrowed to a 1.55 Mb region on chromosome 3, within which we found two potential candidate genes that showed polymorphisms between the mutant and non-mutagenized G1812. Furthermore, the natural variants and elite haplotypes of the two candidates were investigated in a natural population of common wheat. The results showed that the natural variants affect grain yield components, and the dwarf haplotypes show the potential in improving agronomic traits and grain yield. Although the mutation in Rht27 results in severe dwarf phenotype in T. urartu, the natural variants in common wheat showed desirable phenotype, which suggests that Rht27 has the potential to improve wheat yield by utilizing its weak allelic mutation or fine-tuning its expression level.

Genome sequence of the progenitor of wheat A subgenome Triticum urartu.

Triticum urartu (diploid, AA) is the progenitor of the A subgenome of tetraploid (Triticum turgidum, AABB) and hexaploid (Triticum aestivum, AABBDD) wheat1,2. Genomic studies of T. urartu have been useful for investigating the structure, function and evolution of polyploid wheat genomes. Here we report the generation of a high-quality genome sequence of T. urartu by combining bacterial artificial chromosome (BAC)-by-BAC sequencing, single molecule real-time whole-genome shotgun sequencing 3 , linked reads and optical mapping4,5. We assembled seven chromosome-scale pseudomolecules and identified protein-coding genes, and we suggest a model for the evolution of T. urartu chromosomes. Comparative analyses with genomes of other grasses showed gene loss and amplification in the numbers of transposable elements in the T. urartu genome. Population genomics analysis of 147 T. urartu accessions from across the Fertile Crescent showed clustering of three groups, with differences in altitude and biostress, such as powdery mildew disease. The T. urartu genome assembly provides a valuable resource for studying genetic variation in wheat and related grasses, and promises to facilitate the discovery of genes that could be useful for wheat improvement.

Dissection and validation of a promising QTL controlling spikelet number on 5B in bread wheat.

KEY MESSAGE: Five environmentally stable QTLs for spikelet number per spike and days to heading were identified using a high-genetic map containing 95,444 SNPs, among which QSns.ucas-5B was validated using residual heterozygous line at multiple environments. Spikelet number per spike (SNS) and days to heading (DTH) play pivotal roles in the improvement of wheat yield. In this study, a high-density genetic map for a recombinant inbred lines (RILs) population derived from Zhengnong 17 (ZN17) and Yangbaimai (YBM) was constructed using 95,444 single-nucleotide polymorphism (SNP) markers from the Wheat660K SNP array. Our study identified a total of five environmentally stable QTLs for SNS and DTH, one of which was named QSns.ucas-5B, with a physical interval of approximately 545.4-552.1 Mb on the 5BL chromosome arm. Importantly, the elite haplotype within QSns.ucas-5B showed a consistent and positive effect on SNS, grain number and weight per spike, without extending the days to heading. These findings provide a foundation for future efforts to map and clone the gene(s) responsible for QSns.ucas-5B and further indicate the potential application of the developed and validated InDel marker of QSns.ucas-5B for molecular breeding purposes, aimed at improving wheat grain yield.

A Wheat 660 K SNP array-based high-density genetic map facilitates QTL mapping of flag leaf-related traits in wheat.

KEY MESSAGE: A high-density genetic map containing 122,620 SNP markers was constructed, which facilitated the identification of eight major flag leaf-related QTL in relatively narrow intervals. The flag leaf plays an important role in photosynthetic capacity and yield potential in wheat. In this study, we used a recombinant inbred line population containing 188 lines derived from a cross between 'Lankao86' (LK86) and 'Ermangmai' to construct a genetic map using the Wheat 660 K single-nucleotide polymorphism (SNP) array. The high-density genetic map contains 122,620 SNP markers spanning 5185.06 cM. It shows good collinearity with the physical map of Chinese Spring and anchors multiple sequences of previously unplaced scaffolds onto chromosomes. Based on the high-density genetic map, we identified seven, twelve, and eight quantitative trait loci (QTL) for flag leaf length (FLL), width (FLW), and area (FLA) across eight environments, respectively. Among them, three, one, and four QTL for FLL, FLW, and FLA are major and stably express in more than four environments. The physical distance between the flanking markers for QFll.igdb-3B/QFlw.igdb-3B/QFla.igdb-3B is only 444 kb containing eight high confidence genes. These results suggested that we could directly map the candidate genes in a relatively small region by the high-density genetic map constructed with the Wheat 660 K array. Furthermore, the identification of environmentally stable QTL for flag leaf morphology laid a foundation for the following gene cloning and flag leaf morphology improvement.

Fine mapping of the tiller inhibition gene TIN5 in Triticum urartu.

KEY MESSAGE: A tiller inhibition gene TIN5 was delimited to an approximate 2.1 Mb region on chromosome Tu7 that contains 24 annotated genes. Grain yield in wheat (Triticum aestivum L.) is a polygenic trait representing many developmental processes and their interactions with the environments. Among them, tillering capacity is an important agronomic trait for plant architecture and grain yield, but the genetic basis of tiller formation in wheat remains largely unknown. In this study, we identified a tiller inhibition 5 (tin5) mutant from ethyl methane sulfonate treated G1812 (Triticum urartu Thumanjan ex Gandilyan). A mapping population was constructed with tin5/G3146. Based on the sequence differences between G1812 and G3146, large insertions and deletions (≥ 5 bp) were selected and verified, and a skeleton physical map was constructed with genome-wide 168 polymorphic InDel markers. Genetic analysis revealed that the low-tiller phenotype was controlled by a single recessive locus, which we named TIN5. This locus was mapped to a 2.1-Mb region that contained 24 annotated genes on chromosome Tu7. Among these annotated genes, only TuG1812G0700004539 showed a non-synonymous polymorphism between tin5 and the wild type. Our finding will facilitate its map-based cloning and pave the way for an in-depth analysis of the underlying genetic basis of tiller formation and regulation patterns.

Ne2, a typical CC-NBS-LRR-type gene, is responsible for hybrid necrosis in wheat.

Hybrid necrosis, caused by complementary genes Ne1 and Ne2, is a serious barrier for combining desirable traits from different genotypes of wheat, affecting the full utilisation of heterosis. To date, both Ne1 and Ne2 are still not isolated although they were documented decades ago. We report here the map-based cloning and functional characterisation of Ne2, encoding a coiled coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) protein. Homozygous frameshift mutations generated using the CRISPR/Cas9 approach confirmed the Ne2-inducing hybrid necrosis in wheat. Upregulated expression of Ne2 induced by Ne1 and excess hydrogen peroxide accumulation are associated with the necrosis formation. Genetic analyses of a Ne2 allele (Ne2m ) and leaf rust resistance gene LrLC10/Lr13 revealed that they might be the same gene. Furthermore, we demonstrated that the frequency of the Ne2 allele was much lower in landraces (2.00%) compared with that in modern cultivars (13.62%), suggesting that Ne2 allele has been partially applied in wheat genetic improvement. Our findings open opportunities of thoroughly investigating the molecular mechanism of hybrid necrosis, selecting Lr13 and simultaneously avoiding hybrid necrosis in wheat breeding through marker-assisted selection.

Fine mapping of hybrid necrosis gene Ne1 in common wheat (Triticum aestivum L.).

KEY MESSAGE: Hybrid necrosis gene Ne1 was delimited into an approximate 4.06 Mb region on chromosome arm 5BL and an InDel marker that co-segregated with Ne1 alleles was developed. Hybrid necrosis in wheat, characterized by progressive chlorosis and necrosis of plant leaves, tillers or whole plants in certain hybrids, is caused by complementary genes Ne1 and Ne2 located on chromosome arms 5BL and 2BS, respectively. Hybrid necrosis can be a barrier in combining desirable traits from various wheat genotypes. In this study, we fine mapped Ne1 on chromosome arm 5BL, and delimited it to a 4.06 Mb region using large segregating recombinant inbred line families from cross 'Zhengnong 17' × 'Yangbaimai'. Genetic characterization confirmed that the ne1 allele was closely associated with a 2.89 Mb deletion in Zhengnong 17. A tightly linked InDel marker, 5B-InDel385, for Ne1 was developed and was used to predict the presence of Ne1 in a diverse panel of 501 common wheat accessions. Among those accessions, 122 (61%) of 200 landraces were predicted to carry the Ne1 allele, whereas only 79 (26%) of 301 modern cultivars were predicted to carry Ne1. The significant decrease in Ne1 frequency in modern cultivars indicated that the Ne1 allele had been negatively selected in wheat breeding. This study provides a foundation for marker-assisted selection, gene cloning and functional studies of Ne1 in wheat.

Draft genome of the wheat A-genome progenitor Triticum urartu.

Bread wheat (Triticum aestivum, AABBDD) is one of the most widely cultivated and consumed food crops in the world. However, the complex polyploid nature of its genome makes genetic and functional analyses extremely challenging. The A genome, as a basic genome of bread wheat and other polyploid wheats, for example, T. turgidum (AABB), T. timopheevii (AAGG) and T. zhukovskyi (AAGGA(m)A(m)), is central to wheat evolution, domestication and genetic improvement. The progenitor species of the A genome is the diploid wild einkorn wheat T. urartu, which resembles cultivated wheat more extensively than do Aegilops speltoides (the ancestor of the B genome) and Ae. tauschii (the donor of the D genome), especially in the morphology and development of spike and seed. Here we present the generation, assembly and analysis of a whole-genome shotgun draft sequence of the T. urartu genome. We identified protein-coding gene models, performed genome structure analyses and assessed its utility for analysing agronomically important genes and for developing molecular markers. Our T. urartu genome assembly provides a diploid reference for analysis of polyploid wheat genomes and is a valuable resource for the genetic improvement of wheat.

Identification of QTL regions for seedling root traits and their effect on nitrogen use efficiency in wheat (Triticum aestivum L.).

KEY MESSAGE: QTL for a wheat ideotype root system and its plasticity to nitrogen deficiency were characterized. Root system architecture-related traits (RRTs) and their plasticity to nitrogen availability are important for nitrogen acquisition and yield formation in wheat (Triticum aestivum L.). In this study, quantitative trait loci (QTL) analysis was conducted under different nitrogen conditions, using the seedlings of 188 recombinant inbred lines derived from a cross between Kenong 9204 and Jing 411. Fifty-three QTL for seven RRTs and fourteen QTL for the plasticity of these RRTs to nitrogen deficiency were detected. Thirty of these QTL were mapped in nine clusters on chromosomes 2B, 2D, 3A, 3D, 6B, 6D, 7A and 7B. Six of these nine clusters were also colocated with loci for nitrogen use efficiency (NUE)-related traits (NRTs). Among them, three QTL clusters (C2B, C6D and C7B) were highlighted, considering that they individually harbored three stable robust QTL (i.e., QMrl-2B.1, QdRs-6D and QMrl-7B). C2B and C7B stably contributed to the optimal root system, and C6D greatly affected the plasticity of RRTs in response to nitrogen deficiency. However, strong artificial selection was only observed for C7B in 574 derivatives of Kenong 9204. Covariance analysis identified QMrl-7B as the major contributor in C7B that affected the investigated NRTs in mature plants. Phenotypic analysis indicated that thousand kernel weight might represent a "concomitant" above-ground trait of the "hidden" RRTs controlled by C7B, which are used for breeding selection. Dissecting these QTL regions with potential breeding value will ultimately facilitate the selection of donor lines with both high yield and NUE in wheat breeding programs.

SKB1/PRMT5-mediated histone H4R3 dimethylation of Ib subgroup bHLH genes negatively regulates iron homeostasis in Arabidopsis thaliana.

Histone modifications play critical roles in the perception of environmental cues by plants. Here, we report that Shk1 binding protein 1 (SKB1/AtPRMT5), which catalyzes the symmetric dimethylation of histone H4R3 (H4R3sme2), is involved in iron homeostasis in Arabidopsis. The SKB1 lesion mutant exhibited higher iron accumulation in shoots and greater tolerance to iron deficiency than the wild type. The expression of SKB1 was not affected by iron, but the level of H4R3sme2 mediated by SKB1 was related to iron status in plants. We showed by chromatin immunoprecipitation (ChIP) and genome-wide ChIP-seq that SKB1 associated with the chromatin of the Ib subgroup bHLH genes (AtbHLH38, AtbHLH39, AtbHLH100 and AtbHLH101), and symmetrically dimethylated histone H4R3. The quantity of SKB1 that associated with chromatin of the Ib subgroup bHLH genes and the level of H4R3sme2 corresponded to the iron status of plants (higher with increased iron supply and lower when iron was removed). We conclude that SKB1-mediated H4R3sme2 regulates iron homeostasis in Arabidopsis in the context of increasing or decreasing expression of Ib subgroup bHLH genes. Iron deficiency may cause an increase in the disassociation of SKB1 from chromatin of the bHLH genes and a decrease in the level of H4R3sme2, thereby elevating their transcription and enhancing iron uptake. Our findings provide new insight into the molecular mechanisms of iron homeostasis in strategy I plants.

Environmental effects on mineral accumulation in rice grains and identification of ecological specific QTLs.

Optimizing the beneficial mineral elements in rice grains is of interest for rice breeders. To study the environmental effects on mineral accumulation in rice grains, we grew a double-haploid (DH) population derived from the cross between cultivars Chunjiang 06 (CJ06, a japonica rice) and TN1 (an indica rice) under two different ecological environments (Lingshui and Hangzhou, China) and determined the content of Ca, Fe, K, Mg, Mn, P, and Zn in brown rice. These contents show transgressive variation among the DH lines. Subsequently, the quantitative trait loci (QTLs) for mineral accumulation in rice grain were mapped on the chromosomes using CJ06/TN1 population. For the 7 mineral elements investigated, 23 and 9 QTLs were identified for Lingshui and Hangzhou, respectively. Of these, 24 QTLs were reported for the first time in this study and 8 QTLs are consistent with previous reports. Only 2 QTLs for Mg accumulation have been detected in both environments, indicating that mineral accumulation QTLs in rice grains are largely environment dependent. Additionally, co-localizations of QTLs for Mn and Zn, Mg and P, and Mg and Mn accumulation have been observed, implying that these loci might be involved in the accumulation of different elements. Furthermore, the QTLs for the accumulation of Fe, K, Mg, Mn, P, and Zn were mapped to a region close to each other on chromosomes 8 and 9, suggesting that clusters of genes exist on chromosomes 8 and 9. Further characterization of these QTLs will provide a better understanding of the molecular mechanism responsible for mineral accumulation in rice grains.

Comparative genomic and transcriptomic analyses uncover the molecular basis of high nitrogen-use efficiency in the wheat cultivar Kenong 9204.

Studying the regulatory mechanisms that drive nitrogen-use efficiency (NUE) in crops is important for sustainable agriculture and environmental protection. In this study, we generated a high-quality genome assembly for the high-NUE wheat cultivar Kenong 9204 and systematically analyzed genes related to nitrogen uptake and metabolism. By comparative analyses, we found that the high-affinity nitrate transporter gene family had expanded in Triticeae. Further studies showed that subsequent functional differentiation endowed the expanded family members with saline inducibility, providing a genetic basis for improving the adaptability of wheat to nitrogen deficiency in various habitats. To explore the genetic and molecular mechanisms of high NUE, we compared genomic and transcriptomic data from the high-NUE cultivar Kenong 9204 (KN9204) and the low-NUE cultivar Jing 411 and quantified their nitrogen accumulation under high- and low-nitrogen conditions. Compared with Jing 411, KN9204 absorbed significantly more nitrogen at the reproductive stage after shooting and accumulated it in the shoots and seeds. Transcriptome data analysis revealed that nitrogen deficiency clearly suppressed the expression of genes related to cell division in the young spike of Jing 411, whereas this suppression of gene expression was much lower in KN9204. In addition, KN9204 maintained relatively high expression of NPF genes for a longer time than Jing 411 during seed maturity. Physiological and transcriptome data revealed that KN9204 was more tolerant of nitrogen deficiency than Jing 411, especially at the reproductive stage. The high NUE of KN9204 is an integrated effect controlled at different levels. Taken together, our data provide new insights into the molecular mechanisms of NUE and important gene resources for improving wheat cultivars with a higher NUE trait.

Ectopic expression of Lc differentially regulated anthocyanin biosynthesis in the floral parts of tobacco (Nicotiana tobacum L.) plants.

BACKGROUND: Anthocyanins are the conspicuous pigments of flowering plants and participate in several aspects of plant development and defense, such as seeds and pollens dispersal. Leaf colour (Lc) is the first basic/helix-loop-helix (bHLH) transcription factor controlling anthocyanin biosynthesis isolated from maize (Zea mays L.). Ectopic expression of maize Lc enhanced anthocyanin biosynthesis in many plants including tobacco (Nicotiana tobacum L.). However, the molecular regulatory mechanism of anthocyanin biosynthesis in the different floral parts of tobacco remains largely unknown. Therefore, the molecular and biochemical characterization of anthocyanin biosynthesis were investigated in the flowers of both wild type and Lc-transgenic tobacco plants. RESULTS: At the reproductive stage, with respect to the different parts of the flowers in wild type SR1, the calyxes and the pistils were green, and the petals and the filaments showed light pink pigmentation; the Lc-transgenic tobacco exhibited light red in calyxes and crimson in petals and in filaments respectively. Correspondingly, the total anthocyanin contents (TAC) in calyxes, petals and filaments of Lc-transgenic plants were much higher than that of the counterparts in SR1. Though the TAC in anthers of Lc-transgenic plants was low, it was still significantly higher than that of SR1. SR1 has almost the same TAC in the pistils as Lc-transgenic plants. Consistent with the intense phenotype and the increased TAC, Lc was weakly expressed in the calyxes and strongly expressed in petals and filaments of Lc-transgenic plants, while Lc was not detected in SR1. The expression level of NtAN2 in petals was similar between SR1 and Lc-transgenic lines. In agreement with the expression profile of Lc, both early (NtCHS) and late anthocyanin-biosynthetic genes (NtDFR, NtF3'H, and NtANS) were coordinately up-regulated in the counterparts of flowers. HPLC analysis demonstrated that the cyanidin (Cya) deposition was mainly responsible for the intense pigmentation of Lc-transgenic tobacco. CONCLUSIONS: Ectopic expression of Lc greatly enhanced both early- and late- anthocyanin-biosynthetic gene expression, and therefore resulted in the Cya-based TAC increase in the calyxes, the filaments and the petals in tobacco plants.

Sequence-based SSR marker development and their application in defining the Introgressions of LA0716 (Solanum pennellii) in the background of cv. M82 (Solanum lycopersicum).

The introgression lines (ILs) from cv. M82 (Solanum lycopersicum) × LA0716 (S. pennellii) have been proven to be exceptionally useful for genetic analysis and gene cloning. The introgressions were originally defined by RFLP markers at their development. The objectives of this study are to develop polymorphic SSR markers, and to re-define the DNA introgression from LA0716 in the ILs. Tomato sequence data was scanned by software to generate SSR markers. In total, 829 SSRs, which could be robustly amplified by PCR, were developed. Among them, 658 SSRs were dinucleotide repeats, 162 were trinucleotide repeats, and nine were tetranucleotide repeats. The 829 SSRs together with 96 published RFLPs were integrated into the physical linkage map of S. lycopersicum. Introgressions of DNA fragments from LA0716 were re-defined among the 75 ILs using the newly developed SSRs. A specific introgression of DNA fragment from LA0716 was identified in 72 ILs as described previously by RFLP, whereas the specific DNA introgression described previously were not detected in the ILs LA4035, LA4059 and LA4091. The physical location of each investigated DNA introgression was finely determined by SSR mapping. Among the 72 ILs, eight ILs showed a shorter and three ILs (IL3-2, IL12-3 and IL12-3-1) revealed a longer DNA introgression than that framed by RFLPs. Furthermore, 54 previously undefined segments were found in 21 ILs, ranging from 1 to 11 DNA introgressions per IL. Generally, the newly developed SSRs provide additional markers for genetic studies of tomatoes, and the fine definition of DNA introgressions from LA0716 would facilitate the use of the ILs for genetic analysis and gene cloning.

The upregulation of NtAN2 expression at low temperature is required for anthocyanin accumulation in juvenile leaves of Lc-transgenic tobacco (Nicotiana tabacum L.).

Anthocyanins often accumulate in plants subjected to environmental stress, including low temperature. However, the molecular regulatory mechanism of anthocyanin biosynthesis at low temperature is largely unknown. Here, tobacco was transformed with a maize anthocyanin regulatory gene Lc driven by AtSPX3 promoter to investigate the effect of Lc upon the anthocyanin-biosynthesis pathway. We found that the anthocyanin-biosynthesis pathway could not be activated in wild type, while Lc-transgenic tobacco lines exhibited purple pigmentation in juvenile leaves at low temperature. Accordingly, the total anthocyanin contents increased specifically in juvenile leaves in Lc-transgenic lines. Transcriptional analysis showed that NtCHS and NtCHI were induced by low temperature in leaves of wild type and transgenic lines. NtDFR was uniquely expressed in Lc-transgenic lines, but its transcript was not detected in wild type, implying that NtDFR expression in tobacco leaves was dependent on Lc. Furthermore, the expression of NtAN2 (regulatory gene) and NtANS (anthocyanidin synthase gene) was coordinately upregulated in Lc-transgenic lines under low temperature, suggesting that both Lc and NtAN2 might activate the expression of NtANS. Based on our findings and previous reports, we postulated that Lc interacted with NtAN2 induced by low-temperature stress and consequently stimulated anthocyanin biosynthesis in juvenile leaves of Lc-transgenic tobacco lines.