LaDAL1 Coordinates Age and Environmental Signals in the Life Cycle of Larix kaempferi.

Perennial woody plants are long-lived, and their life-cycle events occur in order in each generation, but what drives the occurrence and restart of these events in their offspring is unknown. Based on its age-dependent expression pattern and function, Larix kaempferi DEFICIENS-AGAMOUS-LIKE 1 (LaDAL1), a MADS transcription factor has been suggested to be a time recorder and life-cycle event coordinator. Here, we studied the dynamic spatiotemporal expression pattern of LaDAL1 in the life cycle of L. kaempferi to analyze the molecular mechanism of life-cycle progression. In full view of the life cycle, LaDAL1 transcription was related with life-cycle progression, and its transcript level increased sharply from age 3 to 5 years, which might be the molecular characteristic of the vegetative phase change, and then stayed at a high level. During sexual reproduction, LaDAL1 transcript level decreased sequentially during meiosis and embryogenesis, suggesting that meiosis rapidly lowers the age signal, and after fertilization, the age signal was reset to "0" with the embryogenesis. When a seed germinates, the next generation restarts, and the age is re-counted. Altogether, these results not only provide important and novel insights into the life-cycle progression and transgeneration in perennial woody plants, but also advance our understanding of age recording.

Investigation of the role of AcTPR2 in kiwifruit and its response to Botrytis cinerea infection.

BACKGROUND: Elucidation of the regulatory mechanism of kiwifruit response to gray mold disease caused by Botrytis cinerea can provide the basis for its molecular breeding to impart resistance against this disease. In this study, 'Hongyang' kiwifruit served as the experimental material; the TOPLESS/TOPLESS-RELATED (TPL/TPR) co-repressor gene AcTPR2 was cloned into a pTRV2 vector (AcTPR2-TRV) and the virus-induced gene silencing technique was used to establish the functions of the AcTPR2 gene in kiwifruit resistance to Botrytis cinerea. RESULTS: Virus-induced silencing of AcTPR2 enhanced the susceptibility of kiwifruit to Botrytis cinerea. Defensive enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and phenylalanine ammonia-lyase (PAL) and endogenous phytohormones such as indole acetic acid (IAA), gibberellin (GA3), abscisic acid (ABA), and salicylic acid (SA) were detected. Kiwifruit activated these enzymes and endogenous phytohormones in response to pathogen-induced stress and injury. The expression levels of the IAA signaling genes-AcNIT, AcARF1, and AcARF2-were higher in the AcTPR2-TRV treatment group than in the control. The IAA levels were higher and the rot phenotype was more severe in AcTPR2-TRV kiwifruits than that in the control. These results suggested that AcTPR2 downregulation promotes expression of IAA and IAA signaling genes and accelerates postharvest kiwifruit senescence. Further, Botrytis cinerea dramatically upregulated AcTPR2, indicating that AcTPR2 augments kiwifruit defense against pathogens by downregulating the IAA and IAA signaling genes. CONCLUSIONS: The results of the present study could help clarify the regulatory mechanisms of disease resistance in kiwifruit and furnish genetic resources for molecular breeding of kiwifruit disease resistance.

LaMIR166a-mediated auxin biosynthesis and signalling affect somatic embryogenesis in Larix leptolepis.

Somatic embryogenesis (SE) involves complex molecular signalling pathways. Understanding molecular mechanism of SE in Larix leptolepis (L. leptolepis) can aid research on genetic improvement of gymnosperms. Previously, we obtained five LaMIR166a (miR166a precursor) -overexpression embryonic cell lines in the gymnosperm Larix leptolepis. The proliferation rates of pro-embryogenic masses in transgenic and wild-type lines were calculated. Overexpression of the miR166a precursor LaMIR166a led to slower proliferation. When pro-embryogenic masses were transferred to maturation medium, the relative expression of LaMIR166a and miR166a in the LaMIR166a-overexpression lines was higher than in the wild-type during SE, while LaHDZ31-34 expression levels also increased without negative control by miR166, suggesting that regulation of HD-ZIP III by miR166a exits stage-specific characteristics. The key indole-3-acetic acid (IAA) biosynthetic gene Nitrilase of L. leptolepis (LaNIT) was identified and the effects of miR166a on auxin biosynthesis and signalling genes were studied. During SE, LaNIT, Auxin response factor1 (LaARF1) and LaARF2 mRNA levels and IAA contents were markedly higher in LaMIR166a-overexpression lines, which revealed lower deformity rate of embryos, indicating endogenous IAA synthesis is required for somatic embryo maturation in L. leptolepis. Additionally, the IAA biosynthesis and signalling genes showed similar expression patterns to LaHDZ31-34, suggesting HD-ZIP III genes have a positive regulatory effect on LaNIT. Our results suggest miR166a and LaHDZ31-34 have important roles in auxin biosynthesis and signalling during SE, which might determine if the somatic embryo normally developed to mature in L. leptolepis.

Subcellular Relocalization and Positive Selection Play Key Roles in the Retention of Duplicate Genes of Populus Class III Peroxidase Family.

Gene duplication is the primary source of new genes and novel functions. Over the course of evolution, many duplicate genes lose their function and are eventually removed by deletion. However, some duplicates have persisted and evolved diverse functions. A particular challenge is to understand how this diversity arises and whether positive selection plays a role. In this study, we reconstructed the evolutionary history of the class III peroxidase (PRX) genes from the Populus trichocarpa genome. PRXs are plant-specific enzymes that play important roles in cell wall metabolism and in response to biotic and abiotic stresses. We found that two large tandem-arrayed clusters of PRXs evolved from an ancestral cell wall type PRX to vacuole type, followed by tandem duplications and subsequent functional specification. Substitution models identified seven positively selected sites in the vacuole PRXs. These positively selected sites showed significant effects on the biochemical functions of the enzymes. We also found that positive selection acts more frequently on residues adjacent to, rather than directly at, a critical active site of the enzyme, and on flexible regions rather than on rigid structural elements of the protein. Our study provides new insights into the adaptive molecular evolution of plant enzyme families.

Transcriptome Analysis of mRNA and miRNA in Somatic Embryos of Larix leptolepis Subjected to Hydrogen Treatment.

Hydrogen is a therapeutic antioxidant that has been used extensively in clinical trials. It also acts as a bioactive molecule that can alleviate abiotic stress in plants. However, the biological effects of hydrogen in somatic embryos and the underlying molecular basis remain largely unknown. In this study, the morphological and physiological influence of exogenous H2 treatment during somatic embryogenesis was characterized in Larix leptolepis Gordon. The results showed that exposure to hydrogen increased the proportions of active pro-embryogenic cells and normal somatic embryos. We sequenced mRNA and microRNA (miRNA) libraries to identify global transcriptome changes at different time points during H2 treatment of larch pro-embryogenic masses (PEMs). A total of 45,393 mRNAs and 315 miRNAs were obtained. Among them, 4253 genes and 96 miRNAs were differentially expressed in the hydrogen-treated libraries compared with the control. Further, a large number of the differentially expressed mRNAs and miRNAs were related to reactive oxygen species (ROS) homeostasis and cell cycle regulation. We also identified 4399 potential target genes for 285 of the miRNAs. The differential expression data and the mRNA-miRNA interaction network described here provide new insights into the molecular mechanisms that determine the performance of PEMs exposed to H2 during somatic embryogenesis.

Two NAC transcription factors from Caragana intermedia altered salt tolerance of the transgenic Arabidopsis.

BACKGROUND: Plants are continuously challenged by different environment stresses, and they vary widely in their adjustability. NAC (NAM, ATAF and CUC) transcription factors are known to be crucial in plants tolerance response to abiotic stresses, such as drought and salinity. ANAC019, ANAC055, and ANAC072, belong to the stress-NAC TFs, confer the Arabidopsis abiotic stress tolerance. RESULTS: Here we isolated two stress-responsive NACs, CiNAC3 and CiNAC4, from Caragana intermedia, which were induced by ABA and various abiotic stresses. Localization assays revealed that CiNAC3 and CiNAC4 localized in the nuclei, consistent with their roles as transcription factors. Histochemistry assay using Pro(CiNAC4)::GUS transgenic Arabidopsis showed that the expression of the GUS reporter was observed in many tissues of the transgenic plants, especially in the root vascular system. Overexpression of CiNAC3 and CiNAC4 reduced ABA sensitivity during seed germination, and enhanced salt tolerance of the transgenic Arabidopsis. CONCLUSIONS: We characterised CiNAC3 and CiNAC4 and found that they were induced by numerous abiotic stresses and ABA. GUS histochemical assay of CiNAC4 promoter suggested that root, flower and local damaged tissues were the strongest stained tissues. Overexpression assay revealed that CiNAC4 play essential roles not only in promoting lateral roots formation, but also in responding to salinity and ABA treatment of Arabidopsis.

Reference gene selection for qRT-PCR in Caragana korshinskii Kom. under different stress conditions.

Caragana korshinskii Kom., which is widely distributed in the northwest China and Mongolia, is an important forage bush belonging to the legume family with high economic and ecological value. Strong tolerance ability to various stresses makes C. korshinskii Kom. a valuable species for plant stress research. In this study, suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) were screened from 11 candidate reference genes, including ACT, GAPDH, EF1α, UBQ, TUA, CAP, TUB, TUB3, SKIP1, SKIP5-1 and SKIP5-2. A total of 129 samples under drought, heat, cold, salt, ABA and high pH treatment were profiled, and software such as geNORM, NormFinder and BestKeeper were used for reference gene evaluation and selection. Different suitable reference genes were selected under different stresses. Across all 129 samples, GAPDH, EF1α and SKIP5-1 were found to be the most stable reference genes, and EF1α+SKIP5-1 is the most stable reference gene combination. Conversely, TUA, TUB and SKIP1 were not suitable for using as reference genes owing to their great expression variation under some stress conditions. The relative expression levels of CkWRKY1 were detected using the stable and unstable reference genes and their applicability was confirmed. These results provide some stable reference genes and reference gene combinations for qRT-PCR under different stresses in C. korshinskii Kom. for future research work, and indicate that CkWRKY1 plays essential roles in response to stresses in C. korshinskii.

Overexpression of Larch SCL6 Inhibits Transitions from Vegetative Meristem to Inflorescence and Flower Meristem in Arabidopsis thaliana (L.) Heynh.

SCARECROW-LIKE6 (SCL6) plays a role in the formation and maintenance of the meristem. In Larix kaempferi (Lamb.) Carr., an important afforestation tree species in China, SCL6 (LaSCL6) has two alternative splicing variants-LaSCL6-var1 and LaSCL6-var2-which are regulated by microRNA171. However, their roles are still unclear. In this study, LaSCL6-var1 and LaSCL6-var2 were transformed into the Arabidopsis thaliana (L.) Heynh. genome, and the phenotypic characteristics of transgenic A. thaliana, including the germination percentage, root length, bolting time, flower and silique formation times, inflorescence axis length, and branch and silique numbers, were analyzed to reveal their functions. It was found that LaSCL6-var1 and LaSCL6-var2 overexpression shortened the root length by 41% and 31%, respectively, and increased the inflorescence axis length. Compared with the wild type, the bolting time in transgenic plants was delayed by approximately 2-3 days, the first flower and silique formation times were delayed by approximately 3-4 days, and the last flower and silique formation times were delayed by about 5 days. Overall, the life cycle in transgenic plants was prolonged by approximately 5 days. These results show that LaSCL6 overexpression inhibited the transitions from the vegetative meristem to inflorescence meristem and from the flower meristem to meristem arrest in A. thaliana, revealing the roles of LaSCL6-var1 and LaSCL6-var2 in the fate transition and maintenance of the meristem.

A genome-wide survey of microRNA truncation and 3' nucleotide addition events in larch (Larix leptolepis).

MicroRNAs (miRNAs) play essential roles in numerous developmental and metabolic processes in animals and plants. Although the framework of miRNA biogenesis and function is established, the mechanism of miRNA degradation or modification remains to be investigated in plants. Mature miRNAs may be truncated or added nucleotides to generate variants. A detailed analysis of small RNA deep sequencing data sets resulted in the cloning of a large number of variants derived from larch miRNAs. Many 5'- and/or 3'-end truncated versions of miRNAs suggested that larch miRNAs might be degraded through either 5'-3' or 3'-5'. The relative abundance of variants truncated from 3'-end was higher than that of 5'-end for most miRNAs. The addition of adenine, uridine, and cytidine to the 3'-end of miRNAs was globally present, and the subtle variability in isomiR abundance might be regulated and biologically meaningful. It is the first report for cytidine addition in plant, and our examination of published small RNA deep sequencing data sets of Arabidopsis, rice, and moss suggests that cytidine addition to miRNA 3'-end exists broadly in plants. In addition, the nucleotide addition might be associated with 3'-5' miRNA degradation. Our results provide valuable information for a genome-wide survey of miRNA truncation and modification in larch or plants.

Dynamic expression of small RNA populations in larch (Larix leptolepis).

Small RNAs (sRNAs) are emerging as essential regulators of biological processes. However, several studies have reported that gymnosperms do not express appreciable amounts of 24-nt sRNAs, and conifers in particular may have a unique sRNA-silencing signature. Here, we compared the sRNA transcriptomes of Japanese larch somatic embryos (SE) and seedlings. SE sRNAs exhibited a length bias toward 24 nt, while seedlings showed a bias toward a 21-nt length. We also confirmed that larch is capable of producing 24-nt sRNAs based on a polyacrylamide gel analysis. The sRNA expression patterns varied according to developmental stage, which might be associated with Dicer-like 3 and RNA-dependent RNA polymerase2 (RDR2) levels. Our data suggest that many MIR loci that produce canonical microRNAs (miRNAs, 20-22 nt) and long sRNAs (23-26 nt) have dual functions; the latter were preferentially produced in SE compared to seedlings. However, the ratio of miRNAs to total sRNAs in seedlings was higher than in SE, and most miRNAs were upregulated in seedlings. Trans-acting small interfering RNAs (ta-siRNAs) generated from TAS3 triggered by miR390 were identified, and levels of the three detected ta-siRNAs peaked in mature embryos, which was not consistent with the lowest RDR6 level. These findings indicate that larch, and possibly other gymnosperms, shares a common sRNA pathway with other land plants, and that the sRNA distribution pattern varies according to developmental stage, which may be attributable to the expression of sRNA pathway genes.

Identification of microRNAs in Caragana intermedia by high-throughput sequencing and expression analysis of 12 microRNAs and their targets under salt stress.

KEY MESSAGE: 142 miRNAs were identified and 38 miRNA targets were predicted, 4 of which were validated, in C. intermedia . The expression of 12 miRNAs in salt-stressed leaves was assessed by qRT-PCR. MicroRNAs (miRNAs) are endogenous small RNAs that play important roles in various biological and metabolic processes in plants. Caragana intermedia is an important ecological and economic tree species prominent in the desert environment of west and northwest China. To date, no investigation into C. intermedia miRNAs has been reported. In this study, high-throughput sequencing of small RNAs and analysis of transcriptome data were performed to identify both conserved and novel miRNAs, and also their target mRNA genes in C. intermedia. Based on sequence similarity and hairpin structure prediction, 132 putative conserved miRNAs (12 of which were confirmed to form hairpin precursors) belonging to 31 known miRNA families were identified. Ten novel miRNAs (including the miRNA* sequences of three novel miRNAs) were also discovered. Furthermore, 36 potential target genes of 17 known miRNA families and 2 potential target genes of 1 novel miRNA were predicted; 4 of these were validated by 5' RACE. The expression of 12 miRNAs was validated in different tissues, and these and five target mRNAs were assessed by qRT-PCR after salt treatment. The expression levels of seven miRNAs (cin-miR157a, cin-miR159a, cin-miR165a, cin-miR167b, cin-miR172b, cin-miR390a and cin-miR396a) were upregulated, while cin-miR398a expression was downregulated after salt treatment. The targets of cin-miR157a, cin-miR165a, cin-miR172b and cin-miR396a were downregulated and showed an approximately negative correlation with their corresponding miRNAs under salt treatment. These results would help further understanding of miRNA regulation in response to abiotic stress in C. intermedia.

Genome-wide identification of microRNAs in larch and stage-specific modulation of 11 conserved microRNAs and their targets during somatic embryogenesis.

MicroRNAs (miRNAs) are emerging as essential regulators of biological processes. Somatic embryogenesis is one of the most important techniques for gymnosperm-breeding programs, but there is little understanding of its underlying mechanism. To investigate the roles of miRNAs during somatic embryogenesis in larch, we constructed a small RNA library from somatic embryos. High-throughput sequencing of the library identified 83 conserved miRNAs from 35 families, 16 novel miRNAs, and 14 plausible miRNA candidates, with a high proportion specific to larch or gymnosperms. qRT-PCR analysis demonstrated that both the conserved and novel or candidate miRNAs were expressed in larch. Several miRNA precursor sequences were obtained via RACE. We predicted 110 target genes using bioinformatics, and validated 9 of them by 5' RACE. 11 conserved miRNA families including 17 miRNAs with critical functions in plant development and six target mRNAs were detected by qRT-PCR in the larch SE. Stage-specific expression of miRNAs and their targets indicate their possible modulation on SE of larch: miR171a/b might exert function on PEMs, while miR171c acts in the induction process of larch SE; miR397 and miR398 mainly involved in modulation of PEM propagation and transition to single embryo; miR162 and miR168 exert their regulatory function during total SE process, especially during stages 5-8; miR156, miR159, miR160, miR166, miR167, and miR390 might play regulatory roles during cotyledonary embryo development. These findings indicate that larch and possibly other gymnosperms have complex mechanisms of gene regulation involving specific and common miRNAs operating post-transcriptionally during embryogenesis.

Gene expression profiles of two intraspecific Larix lines and their reciprocal hybrids.

Heterosis has been widely explored in Larix breeding for more than a century, but the molecular mechanisms underlying this phenomenon remain elusive. In the present study, the genome-wide transcript profiles from two Larix genotypes and their reciprocal hybrids were analyzed using Arabidopsis 70-mer oligonucleotide microarrays. Despite sharing the same two parental lines, one of the hybrids showed obvious heterosis, while the other did not. In total, 1,171 genes were differentially expressed between the heterotic hybrid and its parents, of which 133 genes were nonadditive expression. The number of differentially expressed genes between the non-heterotic hybrid and the parents was 939, but only 54 of these genes were nonadditive expression. Further, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses indicated that most of these differentially expressed genes in the heterotic hybrid were associated with several important biological functions such as physiological processes, responses to stimulus, and starch and sucrose metabolism. The reliability of the microarray data was further validated by the Real-time quantitative RT-PCR. A high Pearson linear correlation coefficient value was detected (r = 0.759, P < 0.01). In conclusion, the gene expression profile in the Larix heterotic hybrid was significantly different from that obtained from the non-heterotic hybrid, and more nonadditive differentially expressed genes were detected in the heterotic hybrid, implying that nonadditive effects may be closely associated with the formation of heterosis in the intraspecific Larix hybridization.

Transcriptome profiling and in silico analysis of somatic embryos in Japanese larch (Larix leptolepis).

UNLABELLED: Japanese larch (Larix leptolepis) is an ecologically and economically important species mainly grown in northeastern China, Japan and Europe. However, erratic flowering and poor germplasm resources caused by high embryo abortion rates have hampered breeding of Larix species. Somatic embryogenesis (SE) is an effective tool for the production of L. leptolepis with desirable characteristics, such as expression of totipotency, preparation of synthetic seeds, and genetic transformation. However, public genomic resources for this species are limited. We sequenced 591,759 raw expressed sequence tags (ESTs) from a 454 sequencing cDNA library of L. leptolepis somatic embryos, resulting in 572,403 high-quality reads. These reads were assembled into 70,927 unique sequences (UniGenes), including 32,321 contigs and 38,606 singletons. After removal of low-quality sequences, 65,115 UniGenes were annotated using the UniProtKB program. Based on their sequence similarity with known proteins, the matched 30,372 sequences from 664 species were estimated to represent approximately 19,000 unique genes. Gene ontology analysis revealed 21,324 UniGenes assigned to 51 categories. By Kyoto Encyclopedia of Genes and Genomes mapping, 25,773 transcripts were associated with 160 biochemical pathways. Further analysis screened four signal transduction pathways represented by 337 enzymes and 17 secondary metabolites. In silico analysis reveals that 207 UniESTs in Larix are homologous to MAPKs genes identified from other model plants, which may be involved in regulating SE development. This study provides an initial insight into the Larix transcriptomes of the pro-embryogenic mass and is a sound basis for future studies. KEY MESSAGE: We constructed a large, full-length 454 sequencing cDNA library of Larix leptolepis during somatic embryogenesis. More than 590,000 sequences were obtained and a deep-coverage EST database was constructed.

Integrated transcriptomic and metabolic analyses provide insights into the maintenance of embryogenic potential and the biosynthesis of phenolic acids and flavonoids involving transcription factors in Larix kaempferi (Lamb.) Carr.

Somatic embryogenesis (SE) techniques have been established for micropropagation or basic research related to plant development in many conifer species. The frequent occurrence of non-embryogenic callus (NEC) during SE has impose constraints on the application of somatic embryogenesis SE in Larix kaempferi (Lamb.) Carr, but the potential regulatory mechanisms are poorly understood. In this study, integrated transcriptomic and metabolomic analyses were performed in embryogenic callus (EC) and NEC originating from a single immature zygotic embryo to better decipher the key molecular and metabolic mechanisms required for embryogenic potential maintenance. The results showed that a total of 13,842 differentially expressed genes (DEGs) were found in EC and NEC, among which many were enriched in plant hormone signal transduction, starch and sucrose metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, and the biosynthesis of amino acids pathways. Metabolite profiling showed that 441 differentially accumulated metabolites (DAMs) were identified in EC and NEC. Both EC and NEC had vigorous primary metabolic activities, while most secondary metabolites were upregulated in NEC. Many totipotency-related transcription factor (TF) genes such as BBMs, WUSs, and LEC1 showed higher expression levels in EC compared with NEC, which may result in the higher accumulation of indole 3-acetic acid (IAA) in EC. NEC was characterized by upregulation of genes and metabolites associated with stress responses, such as DEGs involved in jasmonic acid (JA) and ethylene (ETH) biosynthesis and signal transduction pathways, and DEGs and DAMs related to phenylpropanoid and flavonoid biosynthesis. We predicted and analyzed TFs that could target several key co-expressed structural DEGs including two C4H genes, two CcoAOMT genes and three HCT genes involved in phenylpropanoid and flavonoid biosynthesis. Based on the targeted relationship and the co-expression network, two ERFs (Lk23436 and Lk458687), one MYB (Lk34626) and one C2C2-dof (Lk37167) may play an important role in regulating phenolic acid and flavonoid biosynthesis by transcriptionally regulating the expression of these structural genes. This study shows an approach involving integrated transcriptomic and metabolic analyses to obtain insights into molecular events underlying embryogenic potential maintenance and the biosynthesis mechanisms of key metabolites involving TF regulation, which provides valuable information for the improvement of SE efficiency in L. kaempferi.

Melatonin improves the survival of cryopreserved callus of Rhodiola crenulata.

An important aspect of the function of melatonin seems to be the mediation of stress caused by environmental and chemical factors. In the cryopreservation process, environmental changes including osmotic injury, desiccation, and low temperature can impose a series of stresses on plants. In this study, we evaluated the role of melatonin in stress protection during the process of cryopreservation using callus of an endangered plant species Rhodiola crenulata. The survival rate of the cryopreserved callus significantly increased when the callus was pretreated for 5 days with 0.1 µm melatonin prior to freezing in liquid nitrogen. Analysis of antioxidative activity following the pretreatment of callus with 0.1 µm melatonin showed a significant reduction in malondialdehyde production during various steps of cryopreservation. Enhanced peroxidase and catalase activity was observed in the callus after pretreatment with 0.1 µm melatonin compared to the control. These observations provide new evidence of the antioxidant/anti-stress function of melatonin, and it is the first report of its potential application in the preservation of elite endangered germplasm through the process of cryopreservation.

Over-Expression of the Cell-Cycle Gene LaCDKB1;2 Promotes Cell Proliferation and the Formation of Normal Cotyledonary Embryos during Larix kaempferi Somatic Embryogenesis.

Somatic embryogenesis is an effective tool for the production of forest tree seedlings with desirable characteristics; however, the low initiation frequency and productivity of high-quality mature somatic embryos are still limiting factors for Larix kaempferi (Japanese larch). Here, we analyzed the expression pattern of L. kaempferi cyclin-dependent kinase B 1;2 (LaCDKB1;2) during somatic embryogenesis in L. kaempferi and its relationship with the cell proliferation rate. We also analyzed the effect of LaCDKB1;2 over-expression on somatic embryo quality. The results revealed a positive correlation between LaCDKB1;2 expression and the cell proliferation rate during the proliferation stage. After LaCDKB1;2 over-expression, the proliferation rate of cultures increased, and the number of somatic embryos in transgenic cultures was 2.69 times that in non-transformed cultures. Notably, the number of normal cotyledonary embryos in transgenic cultures was 3 times that in non-transformed cultures, indicating that LaCDKB1;2 not only increases the proliferation of cultures and the number of somatic embryos but also improves the quality of somatic embryos. These results provide insight into the regulatory mechanisms of somatic embryogenesis as well as new Larix breeding material.

Concerted control of the LaRAV1-LaCDKB1;3 module by temperature during dormancy release and reactivation of larch.

Dormancy release and reactivation of temperate-zone trees involve the temperature-modulated expression of cell-cycle genes. However, information on the detailed regulatory mechanism is limited. Here, we compared the transcriptomes of the stems of active and dormant larch trees, emphasizing the expression patterns of cell-cycle genes and transcription factors and assessed their relationships and responses to temperatures. Twelve cell-cycle genes and 31 transcription factors were strongly expressed in the active stage. Promoter analysis suggested that these 12 genes might be regulated by transcription factors from 10 families. Altogether, 73 cases of regulation between 16 transcription factors and 12 cell-cycle genes were predicted, while the regulatory interactions between LaMYB20 and LaCYCB1;1, and LaRAV1 and LaCDKB1;3 were confirmed by yeast one-hybrid and dual-luciferase assays. Last, we found that LaRAV1 and LaCDKB1;3 had almost the same expression patterns during dormancy release and reactivation induced naturally or artificially by temperature, indicating that the LaRAV1-LaCDKB1;3 module functions in the temperature-modulated dormancy release and reactivation of larch trees. These results provide new insights into the link between temperature and cell-cycle gene expression, helping to understand the temperature control of tree growth and development in the context of climate change.

Examination of the Transcriptional Response to LaMIR166a Overexpression in Larix kaempferi (Lamb.) Carr.

The study of somatic embryogenesis can provide insight into early plant development. We previously obtained LaMIR166a-overexpressing embryonic cell lines of Larix kaempferi (Lamb.) Carr. To further elucidate the molecular mechanisms associated with miR166 in this species, the transcriptional profiles of wild-type (WT) and three LaMIR166a-overexpressing transgenic cell lines were subjected to RNA sequencing using the Illumina NovaSeq 6000 system. In total, 203,256 unigenes were generated using Trinity de novo assembly, and 2467 differentially expressed genes were obtained by comparing transgenic and WT lines. In addition, we analyzed the cleaved degree of LaMIR166a target genes LaHDZ31-34 in different transgenic cell lines by detecting the expression pattern of LaHdZ31-34, and their cleaved degree in transgenic cell lines was higher than that in WT. The downstream genes of LaHDZ31-34 were identified using Pearson correlation coefficients. Yeast one-hybrid and dual-luciferase report assays revealed that the transcription factors LaHDZ31-34 could bind to the promoters of LaPAP, LaPP1, LaZFP5, and LaPHO1. This is the first report of gene expression changes caused by LaMIR166a overexpression in Japanese larch. These findings lay a foundation for future studies on the regulatory mechanism of miR166.

PAM-less plant genome editing using a CRISPR-SpRY toolbox.

The rapid development of the CRISPR-Cas9, -Cas12a and -Cas12b genome editing systems has greatly fuelled basic and translational plant research1-6. DNA targeting by these Cas nucleases is restricted by their preferred protospacer adjacent motifs (PAMs). The PAM requirement for the most popular Streptococcus pyogenes Cas9 (SpCas9) is NGG (N = A, T, C, G)7, limiting its targeting scope to GC-rich regions. Here, we demonstrate genome editing at relaxed PAM sites in rice (a monocot) and the Dahurian larch (a coniferous tree), using an engineered SpRY Cas9 variant8. Highly efficient targeted mutagenesis can be readily achieved by SpRY at relaxed PAM sites in the Dahurian larch protoplasts and in rice transgenic lines through non-homologous end joining (NHEJ). Furthermore, an SpRY-based cytosine base editor was developed and demonstrated by directed evolution of new herbicide resistant OsALS alleles in rice. Similarly, a highly active SpRY adenine base editor was developed based on ABE8e (ref. 9) and SpRY-ABE8e was able to target relaxed PAM sites in rice plants, achieving up to 79% editing efficiency with high product purity. Thus, the SpRY toolbox breaks a PAM restriction barrier in plant genome engineering by enabling DNA editing in a PAM-less fashion. Evidence was also provided for secondary off-target effects by de novo generated single guide RNAs (sgRNAs) due to SpRY-mediated transfer DNA self-editing, which calls for more sophisticated programmes for designing highly specific sgRNAs when implementing the SpRY genome editing toolbox.