RESUMO
Temperature is a major factor that regulates plant growth and phenotypic diversity. To ensure reproductive success at a range of temperatures, plants must maintain developmental stability of their sexual organs when exposed to temperature fluctuations. However, the mechanisms integrating plant floral organ development and temperature responses are largely unknown. Here, we generated barley and rice loss-of-function mutants in the SEPALLATA-like MADS-box gene MADS8. The mutants in both species form multiple carpels that lack ovules at high ambient temperatures. Tissue-specific markers revealed that HvMADS8 is required to maintain floral meristem determinacy and ovule initiation at high temperatures, and transcriptome analyses confirmed that temperature-dependent differentially expressed genes in Hvmads8 mutants predominantly associate with floral organ and meristem regulation. HvMADS8 temperature-responsive activity relies on increased binding to promoters of downstream targets, as revealed by a cleavage under targets and tagmentation (CUT&Tag) analysis. We also demonstrate that HvMADS8 directly binds to 2 orthologs of D-class floral homeotic genes to activate their expression. Overall, our findings revealed a new, conserved role for MADS8 in maintaining pistil number and ovule initiation in cereal crops, extending the known function of plant MADS-box proteins in floral organ regulation.
Assuntos
Grão Comestível , Genes Homeobox , Grão Comestível/genética , Temperatura , Proteínas de Plantas/metabolismo , Flores/metabolismo , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/metabolismo , Regulação da Expressão Gênica de Plantas/genética , MeristemaRESUMO
One of the challenges for global food security is to reliably and sustainably improve the grain yield of cereal crops. One solution is to modify the architecture of the grain-bearing inflorescence to optimize for grain number and size. Cereal inflorescences are complex structures, with determinacy, branching patterns, and spikelet/floret growth patterns that vary by species. Recent decades have witnessed rapid advancements in our understanding of the genetic regulation of inflorescence architecture in rice, maize, wheat, and barley. Here, we summarize current knowledge on key genetic factors underlying the different inflorescence morphologies of these crops and model plants (Arabidopsis and tomato), focusing particularly on the regulation of inflorescence meristem determinacy and spikelet meristem identity and determinacy. We also discuss strategies to identify and utilize these superior alleles to optimize inflorescence architecture and, ultimately, improve crop grain yield.
Assuntos
Hordeum , Inflorescência , Grão Comestível/genética , Grão Comestível/metabolismo , Poaceae/metabolismo , Hordeum/genética , Triticum/genética , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Meristema , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismoRESUMO
KEY MESSAGE: Genome resequencing uncovers genome-wide DNA polymorphisms that are useful for the development of high-density InDel markers between two barley cultivars. Discovering genomic variations and developing genetic markers are crucial for genetics studies and molecular breeding in cereal crops. Although InDels (insertions and deletions) have become popular because of their abundance and ease of detection, discovery of genome-wide DNA polymorphisms and development of InDel markers in barley have lagged behind other cereal crops such as rice, maize and wheat. In this study, we re-sequenced two barley cultivars, Golden Promise (GP, a classic British spring barley variety) and Hua30 (a Chinese spring barley variety), and mapped clean reads to the reference Morex genome, and identified in total 13,933,145 single nucleotide polymorphisms (SNPs) and 1,240,456 InDels for GP with Morex, 11,297,100 SNPs and 781,687 InDels for Hua30 with Morex, and 13,742,399 SNPs and 1,191,597 InDels for GP with Hua30. We further characterized distinct types, chromosomal distribution patterns, genome location, functional effect, and other features of these DNA polymorphisms. Additionally, we revealed the functional relevance of these identified SNPs/InDels regarding different flowering times between Hua30 and GP within 17 flowering time genes. Furthermore, we developed a series of InDel markers and validated them experimentally in 43 barley core accessions, respectively. Finally, we rebuilt population structure and phylogenetic tree of these 43 barley core accessions. Collectively, all of these genetic resources will facilitate not only the basic research but also applied research in barley.
Assuntos
Hordeum , Hordeum/genética , Genoma de Planta/genética , Filogenia , Mutação INDEL/genética , Polimorfismo de Nucleotídeo Único/genética , DNARESUMO
The timely programmed cell death (PCD) of the tapetum, the innermost somatic anther cell layer in flowering plants, is critical for pollen development, including the deposition and patterning of the pollen wall. Although several genes involved in tapetal PCD and pollen wall development have been characterized, the underlying regulatory mechanism remains elusive. Here we report that PERSISTENT TAPETAL CELL2 (PTC2), which encodes an AT-hook nuclear localized protein in rice (Oryza sativa), is required for normal tapetal PCD and pollen wall development. The mutant ptc2 showed persistent tapetal cells and abnormal pollen wall patterning including absent nexine, collapsed bacula, and disordered tectum. The defective tapetal PCD phenotype of ptc2 was similar to that of a PCD delayed mutant, ptc1, in rice, while the abnormal pollen wall patterning resembled that of a pollen wall defective mutant, Transposable Element Silencing Via AT-Hook, in Arabidopsis (Arabidopsis thaliana). Levels of anther cutin monomers in ptc2 anthers were significantly reduced, as was expression of a series of lipid biosynthetic genes. PTC2 transcript and protein were shown to be present in the anther after meiosis, consistent with the observed phenotype. Based on these data, we propose a model explaining how PTC2 affects anther and pollen development. The characterization of PTC2 in tapetal PCD and pollen wall patterning expands our understanding of the regulatory network of male reproductive development in rice and will aid future breeding approaches.
Assuntos
Apoptose/genética , Flores/crescimento & desenvolvimento , Oryza/crescimento & desenvolvimento , Oryza/genética , Infertilidade das Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/crescimento & desenvolvimento , Motivos AT-Hook/genética , Arabidopsis/genética , Núcleo Celular/metabolismo , Fragmentação do DNA , Flores/genética , Flores/metabolismo , Flores/ultraestrutura , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica de Plantas/genética , Redes Reguladoras de Genes , Genótipo , Metabolismo dos Lipídeos/genética , Lipídeos/análise , Microscopia Eletrônica de Varredura , Mutação , Oryza/metabolismo , Fenótipo , Proteínas de Plantas/genética , Pólen/genética , Pólen/metabolismo , Pólen/ultraestrutura , RNA-Seq , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Clerodendrum inerme (L.) Gaertn, a halophyte, usually grows on coastal beaches as an important mangrove plant. The salt-tolerant mechanisms and related genes of this species that respond to short-term salinity stress are unknown for us. The de novo transcriptome of C. inerme roots was analyzed using next-generation sequencing technology to identify genes involved in salt tolerance and to better understand the response mechanisms of C. inerme to salt stress. RESULTS: Illumina RNA-sequencing was performed on root samples treated with 400 mM NaCl for 0 h, 6 h, 24 h, and 72 h to investigate changes in C. inerme in response to salt stress. The de novo assembly identified 98,968 unigenes. Among these unigenes, 46,085 unigenes were annotated in the NCBI non-redundant protein sequences (NR) database, 34,756 sequences in the Swiss-Prot database and 43,113 unigenes in the evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) database. 52 Gene Ontology (GO) terms and 31 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were matched to those unigenes. Most differentially expressed genes (DEGs) related to the GO terms "single-organism process", "membrane" and "catalytic activity" were significantly enriched while numerous DEGs related to the plant hormone signal transduction pathway were also significantly enriched. The detection of relative expression levels of 9 candidate DEGs by qRT-PCR were basically consistent with fold changes in RNA sequencing analysis, demonstrating that transcriptome data can accurately reflect the response of C. inerme roots to salt stress. CONCLUSIONS: This work revealed that the response of C. inerme roots to saline condition included significant alteration in response of the genes related to plant hormone signaling. Besides, our findings provide numerous salt-tolerant genes for further research to improve the salt tolerance of functional plants and will enhance research on salt-tolerant mechanisms of halophytes.
Assuntos
Clerodendrum/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Estresse Salino/genética , Clerodendrum/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Análise de Sequência de RNARESUMO
BACKGROUND: It is well known that aromatic essential oils extracted from the heartwood of Santalum album L. have wide economic value. However, little is known about the role of terpenoids in response to various adverse environmental stresses as other plants do in the form of signals during plant-environment interactions. RESULTS: In this study, trace amounts of volatiles consisting of α-santalene, epi-ß-santalene, ß-santalene, α-santalol, ß-santalol, (E)-α-bergamotene, (E)-ß-farnesene and ß-bisabolene were found in the leaves of mature S. album trees. We identified more than 40 candidate terpene synthase (TPS) unigenes by mining publicly-available RNA-seq data and characterized the enzymes encoded by three cDNAs: one mono-TPS catalyzes the formation of mostly α-terpineol, and two multifunctional sesqui-TPSs, one of which produces (E)-α-bergamotene and sesquisabinene as major products and another which catalyzes the formation of (E)-ß-farnesene, (E)-nerolidol and (E,E)-farnesol as main products. Metabolite signatures and gene expression studies confirmed that santalol content is closely related with santalene synthase (SaSSY) transcripts in heartwood, which is key enzyme responsible for santalol biosynthesis. However, the expression of three new SaTPS genes differed significantly from SaSSY in the essential oil-producing heartwood. Increased activities of antioxidant enzymes, superoxide dismutase, catalase, peroxidase and ascorbate peroxidase, were detected in different tissues of S. album plants after applying 1 mM methyl jasmonate (MeJA) and 1 mM salicylic acid (SA), or exposure to 4°C, 38°C and high light intensity. MeJA and SA dramatically induced the expression of SaTPS1 and SaTPS2 in leaves. SaTPS1 to 3 transcripts were differentially activated among different tissues under adverse temperature and light stresses. In contrast, almost all SaSSY transcripts decreased in response to these environmental stresses, unlike SaTPS1 to 3. CONCLUSIONS: Multifunctional enzymes were biochemically characterized, including one chloroplastic mono-TPS and two cytosolic sesqui-TPSs in sandalwood. Our results suggest the ecological importance of these three new SaTPS genes in defensive response to biotic attack and abiotic stresses in S. album.
Assuntos
Alquil e Aril Transferases/genética , Proteínas de Plantas/genética , Santalum/fisiologia , Estresse Fisiológico/genética , Acetatos/farmacologia , Alquil e Aril Transferases/metabolismo , Ciclopentanos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Luz , Família Multigênica , Oxilipinas/farmacologia , Filogenia , Folhas de Planta/química , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Ácido Salicílico/farmacologia , Santalum/efeitos dos fármacos , Santalum/genética , Temperatura , Terpenos/análise , Terpenos/química , Terpenos/metabolismo , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/químicaRESUMO
Floral organ shape and size in cereal crops can affect grain size and yield, so genes that regulate their development are promising breeding targets. The lemma, which protects inner floral organs, can physically constrain grain growth; while the awn, a needle-like extension of the lemma, creates photosynthate to developing grain. Although several genes and modules controlling grain size and awn/lemma growth in rice have been characterized, these processes, and the relationships between them, are not well understood for barley and wheat. Here, we demonstrate that the barley E-class gene HvMADS1 positively regulates awn length and lemma width, affecting grain size and weight. Cytological data indicates that HvMADS1 promotes awn and lemma growth by promoting cell proliferation, while multi-omics data reveals that HvMADS1 target genes are associated with cell cycle, phytohormone signaling, and developmental processes. We define two potential targets of HvMADS1 regulation, HvSHI and HvDL, whose knockout mutants mimic awn and/or lemma phenotypes of mads1 mutants. Additionally, we demonstrate that HvMADS1 interacts with APETALA2 (A-class) to synergistically activate downstream genes in awn/lemma development in barley. Notably, we find that MADS1 function remains conserved in wheat, promoting cell proliferation to increase awn length. These findings extend our understanding of MADS1 function in floral organ development and provide insights for Triticeae crop improvement strategies.
Assuntos
Hordeum , Hordeum/genética , Melhoramento Vegetal , Poaceae , Ciclo Celular , Divisão Celular , Grão Comestível , Triticum/genéticaRESUMO
There are typographical errors with the pictures and tables in the original text, and these should be corrected in this corrigendum. The authors state that these corrections do not affect the methods, results, or conclusions of the original text.
RESUMO
The commercial value of Santalum album L. lies in its aromatic heartwood and essential oil. Sesquiterpenes are the main components of sandal essential oil, and these are synthesized through the plant's mevalonate (MVA) and methylerythritol phosphate (MEP) pathways. In this study, the first key rate-limiting enzyme, 1-deoxy-d-xylulose-5-phosphate synthase (SaDXS), was investigated to provide a theoretical molecular basis for the sandalwood MEP sesquiterpene biosynthetic pathway. The biofunctions of SaDXS were also analyzed. SaDXS promoters were successfully cloned from a seven-year-old S. album tree. SaDXS1A/1B promoter activity was verified by a ß-glucuronidase (GUS) assay and by analyzing cis-acting elements of the promoters, which carried light- and methyl jasmonate (MeJA)-responsive signals. In an experiment involving yellow S. album seedlings, exposure to light upregulated SaDXS1A/1B expression and increased chlorophyll and carotenoid contents when overexpressed in Arabidopsis thaliana. Analysis of the expression of SaDXS1A/1B and SaSSy, key genes of santalol biosynthesis, revealed SaDXS1A expression in all tissues whereas SaDXS1B was expressed in tissues that contained photosynthetic pigments, such as stems, leaves and flowers. Sandal seedlings exogenously treated with two hormones, MeJA and ethylene, revealed similar expression patterns for SaDXS1A/1B and SaSSy. Sandal seedlings were treated with an inhibitor of DXS, clomazone, but showed no significant changes in the contents of α-santalene, ß-santalene and α-santalol between treatment and control groups. These results suggest that SaDXS1A/1B play a role in the synthesis of sandalwood sesquiterpenes, providing carbon for downstream secondary metabolites. SaDXS1A/1B also play a role in the biosynthesis of chlorophyll, carotenoids, and primary metabolites.
Assuntos
Óleos Voláteis , Santalum , Sesquiterpenos , Santalum/genética , Santalum/metabolismo , Sesquiterpenos/metabolismo , Óleos Voláteis/metabolismo , Clorofila , Clonagem MolecularRESUMO
Santalum album L., a semi-parasitic evergreen tree, contains economically important essential oil, rich in sesquiterpenoids, such as (Z) α- and (Z) ß-santalol. However, their transcriptional regulations are not clear. Several studies of other plants have shown that basic-helix-loop-helix (bHLH) transcription factors (TFs) were involved in participating in the biosynthesis of sesquiterpene synthase genes. Herein, bHLH TF genes with similar expression patterns and high expression levels were screened by co-expression analysis, and their full-length ORFs were obtained. These bHLH TFs were named SaMYC1, SaMYC3, SaMYC4, SaMYC5, SabHLH1, SabHLH2, SabHLH3, and SabHLH4. All eight TFs had highly conserved bHLH domains and SaMYC1, SaMYC3, SaMYC4, and SaMYC5, also had highly conserved MYC domains. It was indicated that the eight genes belonged to six subfamilies of the bHLH TF family. Among them, SaMYC1 was found in both the nucleus and the cytoplasm, while SaMYC4 was only localized in the cytoplasm and the remaining six TFs were localized in nucleus. In a yeast one-hybrid experiment, we constructed decoy vectors pAbAi-SSy1G-box, pAbAi-CYP2G-box, pAbAi-CYP3G-box, and pAbAi-CYP4G-box, which had been transformed into yeast. We also constructed pGADT7-SaMYC1 and pGADT7-SabHLH1 capture vectors and transformed them into bait strains. Our results showed that SaMYC1 could bind to the G-box of SaSSy, and the SaCYP736A167 promoter, which SaSSy proved has acted as a key enzyme in the synthesis of santalol sesquiterpenes and SaCYP450 catalyzed the ligation of santalol sesquiterpenes into terpene. We have also constructed pGreenII 62-SK-SaMYC1, pGreenII 0800-LUC-SaSSy and pGreenII 0800-LUC-SaCYP736A167 via dual-luciferase fusion expression vectors and transformed them into Nicotiana benthamiana using an Agrobacterium-mediated method. The results showed that SaMYC1 was successfully combined with SaSSy or SaCYP736A167 promoter and the LUC/REN value was 1.85- or 1.55-fold higher, respectively, than that of the control group. Therefore, we inferred that SaMYC1 could activate both SaSSy and SaCYP736A167 promoters.
RESUMO
Sandalwood (Santalum album L.) heartwood-derived essential oil contains a high content of sesquiterpenoids that are economically highly valued and widely used in the fragrance industry. Sesquiterpenoids are biosynthesized via the mevalonate acid and methylerythritol phosphate (MEP) pathways, which are also the sources of precursors for photosynthetic pigments. 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is a secondary rate-limiting enzyme in the MEP pathway. In this paper, the 1416-bp open reading frame of SaDXR and its 897-bp promoter region, which contains putative conserved cis-elements involved in stress responsiveness (HSE and TC-rich repeats), hormone signaling (abscisic acid, gibberellin and salicylic acid) and light responsiveness, were cloned from 7-year-old S. album trees. A bioinformatics analysis suggested that SaDXR encodes a functional and conserved DXR protein. SaDXR was widely expressed in multiple tissues, including roots, twigs, stem sapwood, leaves, flowers, fruit and stem heartwood, displaying significantly higher levels in tissues with photosynthetic pigments, like twigs, leaves and flowers. SaDXR mRNA expression increased in etiolated seedlings exposed to light, and the content of chlorophylls and carotenoids was enhanced in all 35S::SaDXR transgenic Arabidopsis thaliana lines, consistent with the SaDXR expression level. SaDXR was also stimulated by MeJA and H2O2 in seedling roots. α-Santalol content decreased in response to fosmidomycin, a DXR inhibitor. These results suggest that SaDXR plays an important role in the biosynthesis of photosynthetic pigments, shifting the flux to sandalwood-specific sesquiterpenoids.
Assuntos
Aldose-Cetose Isomerases/genética , Santalum/genética , Ácido Abscísico/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Clonagem Molecular/métodos , Flores/genética , Regulação da Expressão Gênica de Plantas/genética , Giberelinas/metabolismo , Complexos Multienzimáticos/genética , Folhas de Planta/genética , Raízes de Plantas/genética , Ácido Salicílico/metabolismo , Santalum/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Sandalwood (Santalum album L.) is famous for its unique fragrance derived from the essential oil of heartwood, whose major components are santalols. To understand the mechanism underlying the biosynthesis of santalols, in this study, we cloned two related genes involved in the mevalonate pathway in S. album coding for acetyl-CoA C-acetyl transferase (AACT) and 3-hydroxy-3-methyglutary-CoA synthase (HMGS). These genes were characterized and functionally analyzed, and their expression profiles were also assessed. An AACT gene designated as SaAACT (GenBank accession No. MH018694) and a HMGS gene designated as SaHMGS (GenBank accession No. MH018695) were successfully cloned from S. album. The deduced SaAACT and SaHMGS proteins contain 415 and 470 amino acids, and the corresponding size of their open-reading frames is 1538 bp and 1807 bp, respectively. Phylogenetic trees showed that the SaAACT protein had the closest relationship with AACT from Hevea brasiliensis and the SaHMGS proteins had the highest homology with HMGS from Siraitia grosvenorii. Functional complementation of SaAACT and SaHMGS in a mutant yeast strain deficient in these proteins confirmed that SaAACT and SaHMGS cDNA encodes functional SaAACT and SaHMGS that mediate mevalonate biosynthesis in yeast. Tissue-specific expression analysis revealed that both genes were constitutively expressed in all examined tissues (roots, sapwood, heartwood, young leaves, mature leaves and shoots) of S. album, both genes showing highest expression in roots. After S. album seedlings were treated with 100 µM methyl jasmonate, the expression levels of SaAACT and SaHMGS genes increased, suggesting that these genes were responsive to this elicitor. These studies provide insight that would allow further analysis of the role of genes related to the sandalwood mevalonate pathway in the regulation of biosynthesis of sandalwood terpenoids and a deeper understanding of the molecular mechanism of santalol biosynthesis.
Assuntos
Acetil-CoA C-Acetiltransferase/genética , Hidroximetilglutaril-CoA Sintase/genética , Proteínas de Plantas/genética , Santalum/genética , Acetil-CoA C-Acetiltransferase/metabolismo , Clonagem Molecular , Hidroximetilglutaril-CoA Sintase/metabolismo , Proteínas de Plantas/metabolismo , Santalum/metabolismoRESUMO
Aux/IAA genes are early auxin-responsive genes and essential for auxin signaling transduction. There is little information about Aux/IAAs in the agriculturally important cereal, barley. Using in silico method, we identified and subsequently characterized 36 Aux/IAAs from the barley genome. Based on their genomic sequences and the phylogenic relationship with Arabidopsis and rice Aux/IAA, the 36 HvIAAs were categorized into two major groups and 14 subgroups. The indication of the presence or absence of these domains for the biological functions and acting mechanisms was discussed. The cis-element distributions in HvIAA promoters suggests that the HvIAAs expressions may not only regulated by auxin (the presence of AuxREs and TGA-element) but also by other hormones and developmental and environmental cues. We then studied the HvIAAs expression in response to NAA (1-Naphthaleneacetic acid) using quantitative real-time PCR (qRT-PCR). Like the promoter analysis, only 14 HvIAAs were upregulated by NAA over two-fold at 4 h. HvIAAs were clustered into three groups based on the spatiotemporal expression data. We confirmed by qRT-PCR that most HvIAAs, especially HvIAA3, HvIAA7, HvIAA8, HvIAA18, HvIAA24 and HvIAA34, are expressed in the developing barley spike compared within seedling, suggesting their roles in regulating spike development. Taken together, our data provide a foundation for further revealing the biological function of these HvIAAs.
Assuntos
Perfilação da Expressão Gênica/métodos , Hordeum/crescimento & desenvolvimento , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Sequenciamento Completo do Genoma/métodos , Simulação por Computador , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hordeum/genética , Hordeum/metabolismo , Ácidos Indolacéticos/farmacologia , Família Multigênica/efeitos dos fármacos , Filogenia , Proteínas de Plantas/metabolismo , Regiões Promotoras GenéticasRESUMO
The GRAS (named after first three identified proteins within this family, GAI, RGA, and SCR) family contains plant-specific genes encoding transcriptional regulators that play a key role in gibberellin (GA) signaling, which regulates plant growth and development. Even though GRAS genes have been characterized in some plant species, little research is known about the GRAS genes in barley (Hordeum vulgare L.). In this study, we observed 62 GRAS members from barley genome, which were grouped into 12 subgroups by using phylogenomic analysis together with the GRAS genes from Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and rice (Oryza sativa). Chromosome localization and gene structure analysis suggested that duplication events and abundant presence of intronless genes might account for the massive expansion of GRAS gene family in barley. The analysis of RNA-seq data indicates the expression pattern of GRAS genes in various tissues at different stages in barley. Noteworthy, our qRT-PCR analysis showed the expression of 18 candidate GRAS genes abundantly in the developing inflorescence, indicating their potential roles in the barley inflorescence development and reproduction. Collectively, our evolutionary and expression analysis of GRAS family are useful for future functional characterization of GA signaling in barley and agricultural improvement.
Assuntos
Evolução Molecular , Giberelinas/metabolismo , Hordeum/genética , Família Multigênica/genética , Arabidopsis/genética , Mapeamento Cromossômico , Sequência Conservada/genética , Regulação da Expressão Gênica de Plantas/genética , Genoma de Planta/genética , Estudo de Associação Genômica Ampla , Oryza/genética , Filogenia , RNA-Seq , Transdução de Sinais/genética , Zea mays/genéticaRESUMO
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used technique to investigate gene expression levels due to its high throughput, specificity, and sensitivity. An appropriate reference gene is essential for RT-qPCR analysis to obtain accurate and reliable results. To date, no reliable reference gene has been validated for the economically tropical tree, sandalwood (Santalum album L.). In this study, 13 candidate reference genes, including 12 novel putative reference genes selected from a large set of S. album transcriptome data, as well as the currently used ß-actin gene (ACT), were validated in different tissues (stem, leaf, root and callus), as well as callus tissue under salicylic acid (SA), jasmonic acid methyl ester (MeJA), and gibberellin (GA) treatments using geNorm, NormFinder, BestKeeper, Delta Ct and comprehensive RefFinder algorithms. Several novel candidate reference genes were much more stable than the currently used traditional gene ACT. ODD paired with Fbp1 for SA treatment, CSA and Fbp3 for MeJA treatment, PP2C and Fbp2 for GA treatment, as well as Fbp1 combined with Fbp2 for the total of three hormone treatments were the most accurate reference genes, respectively. FAB1A, when combined with PP2C, was identified as the most suitable reference gene combination for the four tissues tested, while the combination of HLMt, PPR and FAB1A were the most optimal reference genes for all of the experimental samples. In addition, to verify our results, the relative expression level of the SaSSy gene was evaluated by the validated reference genes and their combinations in the three S. album tissues and under MeJA treatment. The evaluated reference genes in this study will improve the accuracy of RT-qPCR analysis and will benefit S. album functional genomics studies in different tissues and under hormone stimuli in the future.