Transcriptome analysis and functional validation reveal the novel role of LhCYCL in axillary bud development in hybrid Liriodendron.

Shoot branching significantly influences yield and timber quality in woody plants, with hybrid Liriodendron being particularly valuable due to its rapid growth. However, understanding of the mechanisms governing shoot branching in hybrid Liriodendron remains limited. In this study, we systematically examined axillary bud development using morphological and anatomical approaches and selected four distinct developmental stages for an extensive transcriptome analysis. A total of 9,449 differentially expressed genes have been identified, many of which are involved in plant hormone signal transduction pathways. Additionally, we identified several transcription factors downregulated during early axillary bud development, including a noteworthy gene annotated as CYC-like from the TCP TF family, which emerged as a strong candidate for modulating axillary bud development. Quantitative real-time polymerase chain reaction results confirmed the highest expression levels of LhCYCL in hybrid Liriodendron axillary buds, while histochemical ß-glucuronidase staining suggested its potential role in Arabidopsis thaliana leaf axil development. Ectopic expression of LhCYCL in A. thaliana led to an increase of branches and a decrease of plant height, accompanied by altered expression of genes involved in the plant hormone signaling pathways. This indicates the involvement of LhCYCL in regulating shoot branching through plant hormone signaling pathways. In summary, our results emphasize the pivotal role played by LhCYCL in shoot branching, offering insights into the function of the CYC-like gene and establishing a robust foundation for further investigations into the molecular mechanisms governing axillary bud development in hybrid Liriodendron.

A gene regulatory network critical for axillary bud dormancy directly controlled by Arabidopsis BRANCHED1.

The Arabidopsis thaliana transcription factor BRANCHED1 (BRC1) plays a pivotal role in the control of shoot branching as it integrates environmental and endogenous signals that influence axillary bud growth. Despite its remarkable activity as a growth inhibitor, the mechanisms by which BRC1 promotes bud dormancy are largely unknown. We determined the genome-wide BRC1 binding sites in vivo and combined these with transcriptomic data and gene co-expression analyses to identify bona fide BRC1 direct targets. Next, we integrated multi-omics data to infer the BRC1 gene regulatory network (GRN) and used graph theory techniques to find network motifs that control the GRN dynamics. We generated an open online tool to interrogate this network. A group of BRC1 target genes encoding transcription factors (BTFs) orchestrate this intricate transcriptional network enriched in abscisic acid-related components. Promoter::ß-GLUCURONIDASE transgenic lines confirmed that BTFs are expressed in axillary buds. Transient co-expression assays and studies in planta using mutant lines validated the role of BTFs in modulating the GRN and promoting bud dormancy. This knowledge provides access to the developmental mechanisms that regulate shoot branching and helps identify candidate genes to use as tools to adapt plant architecture and crop production to ever-changing environmental conditions.

Brassinosteroid signaling integrates multiple pathways to release apical dominance in tomato.

The control of apical dominance involves auxin, strigolactones (SLs), cytokinins (CKs), and sugars, but the mechanistic controls of this regulatory network are not fully understood. Here, we show that brassinosteroid (BR) promotes bud outgrowth in tomato through the direct transcriptional regulation of BRANCHED1 (BRC1) by the BR signaling component BRASSINAZOLE-RESISTANT1 (BZR1). Attenuated responses to the removal of the apical bud, the inhibition of auxin, SLs or gibberellin synthesis, or treatment with CK and sucrose, were observed in bud outgrowth and the levels of BRC1 transcripts in the BR-deficient or bzr1 mutants. Furthermore, the accumulation of BR and the dephosphorylated form of BZR1 were increased by apical bud removal, inhibition of auxin, and SLs synthesis or treatment with CK and sucrose. These responses were decreased in the DELLA-deficient mutant. In addition, CK accumulation was inhibited by auxin and SLs, and decreased in the DELLA-deficient mutant, but it was increased in response to sucrose treatment. CK promoted BR synthesis in axillary buds through the action of the type-B response regulator, RR10. Our results demonstrate that BR signaling integrates multiple pathways that control shoot branching. Local BR signaling in axillary buds is therefore a potential target for shaping plant architecture.

The GATA factor HANABA TARANU promotes runner formation by regulating axillary bud initiation and outgrowth in cultivated strawberry.

A runner, as an elongated branch, develops from the axillary bud (AXB) in the leaf axil and is crucial for the clonal propagation of cultivated strawberry (Fragaria × ananassa Duch.). Runner formation occurs in at least two steps: AXB initiation and AXB outgrowth. HANABA TARANU (HAN ) encodes a GATA transcription factor that affects AXB initiation in Arabidopsis and promotes branching in grass species, but the underlying mechanism is largely unknown. Here, the function of a strawberry HAN homolog FaHAN in runner formation was characterized. FaHAN transcripts can be detected in the leaf axils. Overexpression (OE) of FaHAN increased the number of runners, mainly by enhancing AXB outgrowth, in strawberry. The expression of the strawberry homolog of BRANCHED1 , a key inhibitor of AXB outgrowth in many plant species, was significantly downregulated in the AXBs of FaHAN -OE lines, whereas the expression of the strawberry homolog of SHOOT MERISTEMLESS, a marker gene for AXB initiation in Arabidopsis, was upregulated. Moreover, several genes of gibberellin biosynthesis and cytokinin signaling pathways were activated, whereas the auxin response pathway genes were repressed. Further assays indicated that FaHAN could be directly activated by FaNAC2, the overexpression of which in strawberry also increased the number of runners. The silencing of FaNAC2 or FaHAN inhibited AXB initiation and led to a higher proportion of dormant AXBs, confirming their roles in the control of runner formation. Taken together, our results revealed a FaNAC2-FaHAN pathway in the control of runner formation and have provided a means to enhance the vegetative propagation of cultivated strawberry.

Genome-wide analysis of MdPLATZ genes and their expression during axillary bud outgrowth in apple (Malus domestica Borkh.).

BACKGROUND: Branching is a plastic character that affects plant architecture and spatial structure. The trait is controlled by a variety of plant hormones through coordination with environmental signals. Plant AT-rich sequence and zinc-binding protein (PLATZ) is a transcription factor that plays an important role in plant growth and development. However, systematic research on the role of the PLATZ family in apple branching has not been conducted previously. RESULTS: In this study, a total of 17 PLATZ genes were identified and characterized from the apple genome. The 83 PLATZ proteins from apple, tomato, Arabidopsis, rice, and maize were classified into three groups based on the topological structure of the phylogenetic tree. The phylogenetic relationships, conserved motifs, gene structure, regulatory cis-acting elements, and microRNAs of the MdPLATZ family members were predicted. Expression analysis revealed that MdPLATZ genes exhibited distinct expression patterns in different tissues. The expression patterns of the MdPLATZ genes were systematically investigated in response to treatments that impact apple branching [thidazuron (TDZ) and decapitation]. The expression of MdPLATZ1, 6, 7, 8, 9, 15, and 16 was regulated during axillary bud outgrowth based on RNA-sequencing data obtained from apple axillary buds treated by decapitation or exogenous TDZ application. Quantitative real-time PCR analysis showed that MdPLATZ6 was strongly downregulated in response to the TDZ and decapitation treatments, however, MdPLATZ15 was significantly upregulated in response to TDZ, but exhibited little response to decapitation. Furthermore, the co-expression network showed that PLATZ might be involved in shoot branching by regulating branching-related genes or mediating cytokinin or auxin pathway. CONCLUSION: The results provide valuable information for further functional investigation of MdPLATZ genes in the control of axillary bud outgrowth in apple.

Lessons from a century of apical dominance research.

The process of apical dominance by which the apical bud/shoot tip of the plant inhibits the outgrowth of axillary buds located below has been studied for more than a century. Different approaches were used over time, with first the physiology era, the genetic era, and then the multidisciplinary era. During the physiology era, auxin was thought of as the master regulator of apical dominance acting indirectly to inhibit bud outgrowth via unknown secondary messenger(s). Potential candidates were cytokinin (CK) and abscisic acid (ABA). The genetic era with the screening of shoot branching mutants in different species revealed the existence of a novel carotenoid-derived branching inhibitor and led to the significant discovery of strigolactones (SLs) as a novel class of plant hormones. The re-discovery of the major role of sugars in apical dominance emerged from modern physiology experiments and involves ongoing work with genetic material affected in sugar signalling. As crops and natural selection rely on the emergent properties of networks such as this branching network, future work should explore the whole network, the details of which are critical but not individually sufficient to solve the 'wicked problems' of sustainable food supply and climate change.

Auxin and CmAP1 regulate the reproductive development of axillary buds in Chinese chestnut (Castanea mollissima).

KEY MESSAGE: Auxin accumulation upregulates the expression of APETALA1 (CmAP1) and subsequently activates inflorescence primordium development in axillary buds of chestnut. The architecture of fruiting branches is a key determinant of chestnut yield. Normally, axillary buds at the top of mother fruiting branches develop into flowering shoots and bear fruits, and the lower axillary buds develop into vegetative shoots. Decapitation of the upper axillary buds induces the lower buds to develop into flowering shoots. How decapitation modulates the tradeoff between vegetative and reproductive development is unclear. We detected inflorescence primordia within both upper and lower axillary buds on mother fruiting branches. The level of the phytohormones 3-indoleacetic acid (IAA) and trans-zeatin (tZ) increased in the lower axillary buds in response to decapitation. Exogenous application of the synthetic analogues 1-naphthylacetic acid (NAA) or 6-benzyladenine (6-BA) blocked or promoted, respectively, the development of the inflorescence primordia in axillary buds. The transcript levels of the floral identity gene CmAP1 increased in axillary buds following decapitation. An auxin response element TGA-box is present in the CmAP1 promoter and influenced the CmAP1 promoter-driven expression of ß-glucuronidase (GUS) in floral organs in Arabidopsis, suggesting that CmAP1 is induced by auxin. We propose that decapitation releases axillary bud outgrowth from inhibition caused by apical dominance. During this process, decapitation-induced accumulation of auxin induces CmAP1 expression, subsequently promoting the reproductive development of axillary buds.

Physiological changes besides the enhancement of pigmentation in Petunia hybrida caused by overexpression of PhAN2, an R2R3-MYB transcription factor.

KEY MESSAGE: Ectopic expression of PhAN2 in vegetative tissue can improve regeneration and adventitious rooting but inhibit axillary bud outgrowth of petunia, while overexpression specifically in flowers could shorten longevity. Anthocyanin 2 has been only treated as a critical positive regulation factor of anthocyanin biosynthesis in petunia flowers. To determine if this gene had other functions in plant growth, we overexpressed this gene in an an2 mutant petunia cultivar driven by promoters with different strengths or tissue specificity. Various physiological processes of transformants in different growth stages and environments were analyzed. Besides the expected pigmentation improvement in different tissues, the results also showed that ectopic expression of AN2 could improve the regeneration skill but inhibit the axillary bud germination of in vitro plants. Moreover, the rooting ability of shoot tips of transformants was significantly improved, while some transgenic lines' flower longevity was shortened. Gene expression analysis showed that the transcripts level of AN2, partner genes anthocyanin 1 (AN1), anthocyanin 11 (AN11), and target gene dihydroflavonol 4-reductase (DFR) was altered in the different transgenic lines. In addition, ethylene biosynthesis-related genes 1-aminocyclopropane-1-carboxylic acid synthase (ACS1) and ACC oxidase (ACO1) were upregulated in rooting and flower senescence processes but at different time points. Overall, our data demonstrate that the critical role of this AN2 gene in plant growth physiology may extend beyond that of a single activator of anthocyanin biosynthesis.

The role of strigolactone analog (GR24) in endogenous hormone metabolism and hormone-related gene expression in tobacco axillary buds.

KEY MESSAGE: Strigolactone has the potential to influence hormone metabolism, in addition to having a role in inhibiting axillary bud elongation, which could be regulated by the expression of phytohormones-related genes. The elongation of axillary buds affects the economic benefits of tobacco. In this study, it was investigated the effect of strigolactone (SL) on the elongation of tobacco axillary buds and its endogenous hormone metabolism and related gene expression by applying the artificial analog of SL, GR24, and an inhibitor of SL synthesis, TIS-108, to the axillary buds. The results showed that the elongation of axillary buds was significantly inhibited by GR24 on day 2 and day 9. Ultra-high-performance liquid-chromatography-mass spectrometry results further showed that SL significantly affected the metabolism of endogenous plant hormones, altering both their levels and the ratios between each endogenous hormone. Particularly, the levels of auxin (IAA), trans-zeatin-riboside (tZR), N6-(∆2-isopentenyl) adenine (iP), gibberellin A4 (GA4), jasmonic acid (JA), and jasmonoyl isoleucine (JA-Ile) were decreased after GR24 treatment on day 9, but the levels of 1-aminocyclopropane-1-carboxylic acid (ACC) and gibberellin A1 (GA1) were significantly increased. Further analysis of endogenous hormonal balance revealed that after the treatment with GR24 on day 9, the ratio of IAA to cytokinin (CTK) was markedly increased, but the ratios of IAA to abscisic acid (ABA), salicylic acid (SA), ACC, JAs, and, GAs were notably decreased. In addition, according to RNA-seq analysis, multiple differentially expressed genes were found, such as GH3.1, AUX/IAA, SUAR20, IPT, CKX1, GA2ox1, ACO3, ERF1, PR1, and HCT, which may play critical roles in the biosynthesis, deactivation, signaling pathway of phytohormones, and the biosynthesis of flavonoids to regulate the elongation of axillary buds in tobacco. This work lays the certain theoretical foundation for the application of SL in regulating the elongation of axillary buds of tobacco.

KNOX Genes Were Involved in Regulating Axillary Bud Formation of Chrysanthemum × morifolium.

Branching is an important agronomic and economic trait in cut chrysanthemums. The axillary meristem (AM) formation of the axillary buds of cut chrysanthemums has a decisive role in its branching characteristics. However, little is known about the regulation mechanism of axillary meristem formation in chrysanthemums at the molecular level. Members of the Homeobox gene family especially genes belonging to the class I KNOX branch play a key role in regulating the axillary bud growth and development processes of plants. In this study, three genes belonging to the class I KNOX branch, CmKNAT1, CmKNAT6, and CmSTM were cloned from chrysanthemums, and their functions in regulating axillary bud formation were examined. The subcellular localization test showed that these three KNOX genes were expressed in the nucleus, so all of them might function as transcription factors. The results of the expression profile analysis showed that these three KNOX genes were highly expressed in the AM formation stage of axillary buds. Overexpression of KNOX genes result in a wrinkled leaf phenotype in tobacco and Arabidopsis, which may be related to the excessive division of leaf cells, resulting in the proliferation of leaf tissue. Furthermore, overexpression of these three KNOX genes enhances the regeneration ability of tobacco leaves, indicating that these three KNOX genes may participate in the regulation of cell meristematic ability, thus promoting the formation of buds. In addition, the results of fluorescence quantitative testing showed that these three KNOX genes may promote the formation of chrysanthemum axillary buds by promoting the cytokinin pathway while inhibiting the auxin and gibberellin pathways. In conclusion, this study demonstrated that CmKNAT1, CmKNAT6, and CmSTM genes were involved in regulating axillary bud formation of Chrysanthemum × morifolium and preliminarily revealed the molecular mechanism of their regulation of AM formation. These findings may provide a theoretical basis and candidate gene resources for genetic engineering breeding of new varieties of cut chrysanthemums without lateral branches.

Polyploidization-enhanced effective clonal reproduction endows the successful invasion of Solidago canadensis.

Clonality and ploidy levels are positively associated with plant invasiveness. However, there is still no consensus on whether polyploidization can promote the invasion of alien plants by enhancing clonality. Our recent long-term community succession study found that the more vigorous clone of introduced polyploid Solidago canadensis succeeded into mono-dominant community, which seems to be a positive correlationship between polyploidization and clonal reproduction. However, the formation process of clonal ramet and how polyploidization improves the clonal reproduction of S. canadensis remains unknown. Here, we compared clonal growth ability among diploids and polyploids of S. canadensis from native and introduced ranges in a common garden. Results showed that the rhizomes of S. canadensis originated from axillary buds of dense nodes at the basal stem of seedling and then produced into clonal ramets from the rhizomes. Diploids had denser nodes and more buds, developed more rhizomes per unit mass and produced more clonal propagules at the early growth stage compared with polyploids. However, the number of juvenile and secondary rhizomes, as well as the diameter and length of rhizomes in polyploid populations was significantly higher or greater than those of diploids, and those clonal traits in introduced polyploids were significantly higher than in native polyploids. Moreover, a phalanx growth form was observed in native and introduced diploid populations, which allocated about 3% and 5% of the total biomass to rhizomes, respectively, resulting in short and weak rhizomes. However, native and introduced polyploids allocated about 35% and 40%, respectively, of the total biomass to rhizomes, resulting in long and strong rhizomes, which were guerrilla growth forms. This study firstly shows that polyploidization enhanced the effective clonal reproduction of S. canadensis through pre-adaptation and rapid post-adaptation evolution, and consequently contributed to its successful invasion.

The branch-thorn occurrence of Lycium ruthenicum is associated with leaf DNA hypermethylation in response to soil water content.

BACKGROUND: Lycium ruthenicum is an eco-economic shrub which can exist in two forms, thorny and thornless under varying soil moisture conditions. The aim of this study was to determine if the two forms of L. ruthenicum were influenced by soil water content (SWC) and to test the three-way link among SWC, occurrence of branch-thorn and DNA methylation modification. METHODS AND RESULTS: Here, pot experiment was carried out to reveal the influence of SWC on the occurrence of branch-thorn and then paraffin sections, scanning electron microscope and methylation-sensitive amplification polymorphism(MSAP) analysis were used to determine the three-way link among SWC, branch-thorn occurrence and DNA methylation. The results showed that (a) soil drought promoted the development of thorn primordium into branch-thorn and (b) branch-thorn covered axillary bud to protect it against drought and other stresses; (c) the branch-thorn occurrence response to drought was correlated with hypermethylation of CCGG sites and (d) thorny and thornless plants of a clone were distinguished successfully based on the MSAP profiles of their leaves. CONCLUSIONS: Branch-thorns of the L. ruthenicum clone, which occurred in response to drought, covered axillary buds to protect them against drought and other stresses; thorn primordium of the clone did not develop into branch-thorn under the adequate soil moisture condition. The occurrence and absence of the branch-thorns were correlated with the hyper- and hypo-methylation, respectively. We proposed that the branch-thorn plasticity might be an adjustment strategy for the environment, which seems to support the theory of "Use in, waste out".

CsBRC1 inhibits axillary bud outgrowth by directly repressing the auxin efflux carrier CsPIN3 in cucumber.

Shoot branching is an important agronomic trait that directly determines plant architecture and affects crop productivity. To promote crop yield and quality, axillary branches need to be manually removed during cucumber production for fresh market and thus are undesirable. Auxin is well known as the primary signal imposing for apical dominance and acts as a repressor for lateral bud outgrowth indirectly. The TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family gene BRANCHED1 (BRC1) has been shown to be the central integrator for multiple environmental and developmental factors that functions locally to inhibit shoot branching. However, the direct molecular link between auxin and BRC1 remains elusive. Here we find that cucumber BRANCHED1 (CsBRC1) is expressed in axillary buds and displays a higher expression level in cultivated cucumber than in its wild ancestor. Knockdown of CsBRC1 by RNAi leads to increased bud outgrowth and reduced auxin accumulation in buds. We further show that CsBRC1 directly binds to the auxin efflux carrier PIN-FORMED (CsPIN3) and negatively regulates its expression in vitro and in vivo. Elevated expression of CsPIN3 driven by the CsBRC1 promoter results in highly branched cucumber with decreased auxin levels in lateral buds. Therefore, our data suggest that CsBRC1 inhibits lateral bud outgrowth by direct suppression of CsPIN3 functioning and thus auxin accumulation in axillary buds in cucumber, providing a strategy to breed for cultivars with varying degrees of shoot branching grown in different cucumber production systems.

Exogenous phytohormone application and transcriptome analysis of Mikania micrantha provides insights for a potential control strategy.

Effective and complete control of the invasive weed Mikania micrantha is required to avoid increasing damages. We exogenously applied indole 3-acetic acid (IAA), gibberellin (GA), and N-(2-Chloro-4-pyridyl)-N'-phenylurea (CPPU), and their combinations i.e. IAA + CPPU (IC), GA + CPPU (GC), and GA + IAA + CPPU (GIC), at 5, 10, 25, 50, and 75 ppm against distilled water as a control (CK), to examine their effects on the weed. The increasing concentrations of these hormones when applied alone or in combination were fatal to M. micrantha and led towards the death of inflorescences and/or florets. CPPU and GIC were found as the most effective phytohormones. Transcriptome analysis revealed differential regulation of genes in auxin, cytokinin, gibberellin and abscisic acid signaling pathways, suggesting their role in the prohibition of axillary bud differentiation. Collectively, CPPU and GIC at a high concentration (75 ppm) could be used as a control measure to protect forests and other lands from the invasion of M. micrantha.

Phytoplasma Infection Blocks Starch Breakdown and Triggers Chloroplast Degradation, Leading to Premature Leaf Senescence, Sucrose Reallocation, and Spatiotemporal Redistribution of Phytohormones.

Witches'-broom (WB, excessive initiation, and outgrowth of axillary buds) is one of the remarkable symptoms in plants caused by phytoplasmas, minute wall-less intracellular bacteria. In healthy plants, axillary bud initiation and outgrowth are regulated by an intricate interplay of nutrients (such as sugars), hormones, and environmental factors. However, how these factors are involved in the induction of WB by phytoplasma is poorly understood. We postulated that the WB symptom is a manifestation of the pathologically induced redistribution of sugar and phytohormones. Employing potato purple top phytoplasma and its alternative host tomato (Solanum lycopersicum), sugar metabolism and transportation, and the spatiotemporal distribution of phytohormones were investigated. A transmission electron microscopy (TEM) analysis revealed that starch breakdown was inhibited, resulting in the degradation of damaged chloroplasts, and in turn, premature leaf senescence. In the infected source leaves, two marker genes encoding asparagine synthetase (Sl-ASN) and trehalose-6-phosphate synthase (Sl-TPS) that induce early leaf senescence were significantly up-regulated. However, the key gibberellin biosynthesis gene that encodes ent-kaurene synthase (Sl-KS) was suppressed. The assessment of sugar content in various infected tissues (mature leaves, stems, roots, and leaf axils) indicated that sucrose transportation through phloem was impeded, leading to sucrose reallocation into the leaf axils. Excessive callose deposition and the resulting reduction in sieve pore size revealed by aniline blue staining and TEM provided additional evidence to support impaired sugar transport. In addition, a spatiotemporal distribution study of cytokinin and auxin using reporter lines detected a cytokinin signal in leaf axils where the axillary buds initiated. However, the auxin responsive signal was rarely present in such leaf axils, but at the tips of the newly elongated buds. These results suggested that redistributed sucrose as well as cytokinin in leaf axils triggered the axillary bud initiation, and auxin played a role in the bud elongation. The expression profiles of genes encoding squamosa promoter-binding proteins (Sl-SBP1), and BRANCHED1 (Sl-BRC1a and Sl-BRC1b) that control axillary bud release, as determined by quantitative reverse transcription (qRT)-PCR, indicated their roles in WB induction. However, their interactions with sugars and cytokinins require further study. Our findings provide a comprehensive insight into the mechanisms by which phytoplasmas induce WB along with leaf chlorosis, little leaf, and stunted growth.

Transcriptome Analysis Reveals Endogenous Hormone Changes during Spike Development in Phalaenopsis.

Phalaenopsis orchids are popular worldwide due to their high ornamental and economic value; the spike and inflorescence formation of their flowers could be efficiently controlled under proper conditions. In this study, transcriptomic profiles and endogenous hormone changes were investigated to better understand the spike formation of Phalaenopsis. Morphological observations revealed four spike initiation statuses (i.e., S0: the status refers to axillary buds remaining dormant in the leaf axils; S1: the status refers to the 0.5 cm-long initial spike; S2: the status refers to the 1 cm-long spike; S3: the status refers to the 3 cm-long spike) during the process of spike development, while anatomical observations revealed four related statuses of inflorescence primordium differentiation. A total of 4080 differentially expressed genes were identified based on pairwise comparisons of the transcriptomic data obtained from the S0 to S3 samples; high levels of differential gene expression were mostly observed in S1 vs. S2, followed by S0 vs. S1. Then, the contents of 12 endogenous hormones (e.g., irindole-3-acetic acid (IAA), salicylic acid (SA), abscisic acid (ABA), gibberellins, and cytokinins) were measured. The results showed that the ABA content was decreased from S0 to S1, while the gibberellic acid 1 (GA1) content exhibited an opposite trend, indicating the reduction in ABA levels combined with the increase in GA1 levels in S0 promoted the axillary bud dormancy breaking, preparing for the following spike initiation. The GA20 oxidase and ABA 8'-hydroxylase genes, which are involved in endogenous hormone metabolism and signaling pathways, displayed similar expression patterns, suggesting they were probably the key genes participating in the GA and ABA regulation. Taken together, the findings of this study indicate that GA and ABA may be the key endogenous hormones breaking the dormancy and promoting the germination of axillary buds in Phalaenopsis.

Genome-wide identification of the tobacco GDSL family and apical meristem-specific expression conferred by the GDSL promoter.

BACKGROUND: GDSL esterases/lipases are a large protein subfamily defined by the distinct GDSL motif, and play important roles in plant development and stress responses. However, few studies have reported on the role of GDSLs in the growth and development of axillary buds. This work aims to identify the GDSL family members in tobacco and explore whether the NtGDSL gene contributes to development of the axillary bud in tobacco. RESULTS: One hundred fifty-nine GDSL esterase/lipase genes from cultivated tobacco (Nicotiana tabacum) were identified, and the dynamic changes in the expression levels of 93 of these genes in response to topping, as assessed using transcriptome data of topping-induced axillary shoots, were analysed. In total, 13 GDSL esterase/lipase genes responded with changes in expression level. To identify genes and promoters that drive the tissue-specific expression in tobacco apical and axillary buds, the expression patterns of these 13 genes were verified using qRT-PCR. GUS activity and a lethal gene expression pattern driven by the NtGDSL127 promoter in transgenic tobacco demonstrated that NtGDSL127 is specifically expressed in apical buds, axillary buds, and flowers. Three separate deletions in the NtGDSL127 promoter demonstrated that a minimum upstream segment of 235 bp from the translation start site can drive the tissue-specific expression in the apical meristem. Additionally, NtGDSL127 responded to phytohormones, providing strategies for improving tobacco breeding and growth. CONCLUSION: We propose that in tobacco, the NtGDSL127 promoter directs expression specifically in the apical meristem and that expression is closely correlated with axillary bud development.

A novel adenylate isopentenyltransferase 5 regulates shoot branching via the ATTTA motif in Camellia sinensis.

BACKGROUND: Shoot branching is one of the important agronomic traits affecting yields and quality of tea plant (Camellia sinensis). Cytokinins (CTKs) play critical roles in regulating shoot branching. However, whether and how differently alternative splicing (AS) variant of CTKs-related genes can influence shoot branching of tea plant is still not fully elucidated. RESULTS: In this study, five AS variants of CTK biosynthetic gene adenylate isopentenyltransferase (CsA-IPT5) with different 3' untranslated region (3' UTR) and 5' UTR from tea plant were cloned and investigated for their regulatory effects. Transient expression assays showed that there were significant negative correlations between CsA-IPT5 protein expression, mRNA expression of CsA-IPT5 AS variants and the number of ATTTA motifs, respectively. Shoot branching processes induced by exogenous 6-BA or pruning were studied, where CsA-IPT5 was demonstrated to regulate protein synthesis of CsA-IPT5, as well as the biosynthesis of trans-zeatin (tZ)- and isopentenyladenine (iP)-CTKs, through transcriptionally changing ratios of its five AS variants in these processes. Furthermore, the 3' UTR AS variant 2 (3AS2) might act as the predominant AS transcript. CONCLUSIONS: Together, our results indicate that 3AS2 of the CsA-IPT5 gene is potential in regulating shoot branching of tea plant and provides a gene resource for improving the plant-type of woody plants.

Improved node culture methods for rapid vegetative propagation of switchgrass (Panicum virgatum L.).

BACKGROUND: Switchgrass (Panicum virgatum L.) is an important bioenergy and forage crop. The outcrossing nature of switchgrass makes it infeasible to maintain a genotype through sexual propagation. Current asexual propagation protocols in switchgrass have various limitations. An easy and highly-efficient vegetative propagation method is needed to propagate large natural collections of switchgrass genotypes for genome-wide association studies (GWAS). RESULTS: Micropropagation by node culture was found to be a rapid method for vegetative propagation of switchgrass. Bacterial and fungal contamination during node culture is a major cause for cultural failure. Adding the biocide, Plant Preservative Mixture (PPM, 0.2%), and the fungicide, Benomyl (5 mg/l), in the incubation solution after surface sterilization and in the culture medium significantly decreased bacterial and fungal contamination. In addition, "shoot trimming" before subculture had a positive effect on shoot multiplication for most genotypes tested. Using the optimized node culture procedure, we successfully propagated 330 genotypes from a switchgrass GWAS panel in three separate experiments. Large variations in shoot induction efficiency and shoot growth were observed among genotypes. Separately, we developed an in planta node culture method by stimulating the growth of aerial axillary buds into shoots directly on the parent plants, through which rooted plants can be generated within 6 weeks. By circumventing the tissue culture step and avoiding application of exterior hormones, the in planta node culture method is labor- and cost-efficient, easy to master, and has a high success rate. Plants generated by the in planta node culture method are similar to seedlings and can be used directly for various experiments. CONCLUSIONS: In this study, we optimized a switchgrass node culture protocol by minimizing bacterial and fungal contamination and increasing shoot multiplication. With this improved protocol, we successfully propagated three quarters of the genotypes in a diverse switchgrass GWAS panel. Furthermore, we established a novel and high-throughput in planta node culture method. Together, these methods provide better options for researchers to accelerate vegetative propagation of switchgrass.

Correlation analysis of the transcriptome and metabolome reveals the role of the flavonoid biosynthesis pathway in regulating axillary buds in upland cotton (Gossypium hirsutum L.).

MAIN CONCLUSION: Flavonoids are involved in axillary bud development in upland cotton. The phenylpropanoid and flavonoid biosynthesis pathways regulate axillary bud growth by promoting the transport of auxin in upland cotton. In cotton production, simplified cultivation and mechanical harvesting are emerging trends that depend on whether the cotton plant type meets production requirements. The axillary bud is an important index of cotton plant-type traits, and the molecular mechanism of axillary bud development in upland cotton has not yet been completely studied. Here, a combined investigation of transcriptome and metabolome analyses in G. hirsutum CCRI 117 at the fourth week (stage 1), fifth week (stage 2) and sixth week (stage 3) after seedling emergence was performed. The metabolome results showed that the total lipid, amino acid and organic acid contents in the first stalk node decreased during axillary bud development. The abundance of 71 metabolites was altered between stage 2 and stage 1, and 32 metabolites exhibited significantly altered abundance between stage 3 and stage 2. According to the correlation analysis of metabolome and transcriptome profiles, we found that phenylpropanoid and flavonoid biosynthesis pathways exhibit high enrichment degrees of both differential metabolites and differential genes in three stages. Based on the verification of hormone, soluble sugar and flavonoid detection, we propose a model for flavonoid-mediated regulation of axillary bud development in upland cotton, revealing that the decrease in secondary metabolites of phenylpropanoid and flavonoid biosynthesis is an essential factor to promote the transport of auxin and subsequently promote the growth of axillary buds. Our findings provide novel insights into the regulation of phenylpropanoid and flavonoid biosynthesis in axillary bud development and could prove useful for cultivating machine-harvested cotton varieties with low axillary buds.