Rapid analysis of strigolactone receptor activity in a Nicotiana benthamiana dwarf14 mutant

DWARF14 (D14) is an ɑ/β-hydrolase and receptor for the plant hormone strigolactone (SL) in angiosperms. Upon SL perception, D14 works with MORE AXILLARY GROWTH2 (MAX2) to trigger polyubiquitination and degradation of DWARF53(D53)-type proteins in the SUPPRESSOR OF MAX2 1-LIKE (SMXL) family. We used CRISPR-Cas9 to generate knockout alleles of the two homoeologous D14 genes in the Nicotiana benthamiana genome. The Nbd14a,b double mutant had several phenotypes that are consistent with the loss of SL perception in other plants, including increased axillary bud outgrowth, reduced height, shortened petioles, and smaller leaves. A ratiometric fluorescent reporter system was used to monitor degradation of SMXL7 from Arabidopsis thaliana (AtSMXL7) after transient expression in N. benthamiana and treatment with the strigolactone analog GR24. AtSMXL7 was degraded after treatment with GR245DS, which has the stereochemical configuration of SLs, as well as its enantiomer GR24ent-5DS. In Nbd14a,b leaves, AtSMXL7 abundance was unaffected by GR24. Transient coexpression of AtD14 with the AtSMXL7 reporter in Nbd14a,b restored the degradation response to GR24, but required an active catalytic triad. With this platform, we evaluated the ability of several AtD14 mutants that had not been characterized in plants to target AtSMXL7 for degradation.

Metabolite Profiles of Energy Cane and Sugarcane Reveal Different Strategies During the Axillary Bud Outgrowth

Sugarcane (Saccharum spp.) is one of the most well-known plants which possesses a large accumulation of sucrose. Another cultivar, energy cane, is an interspecific hybrid with higher fiber and lower sugar content than sugarcane. Commercial cultivation of sugarcane and energy cane is carried out by vegetative propagation, through the distribution of culm segments (setts) or pre-sprouted seedlings (PSS). In this context, the metabolism of axillary bud outgrowth is crucial for cultures that use vegetative propagation. In this work, we evaluate the metabolic profile of sugarcane and energy cane in the early hours during the axillary bud outgrowth. Sugarcane showed few metabolic changes, except for the significant increase in glutamate levels, which may be associated with root formation in the culm. In contrast, energy cane presented significant changes in amino acid catabolism, increased levels of reducing sugars, lipids, and metabolite activity in the phenylpropanoid pathway. These results together reveal changes in the energy and redox status of the cell, electron transport for the TCA cycle, and an increase in compounds related to cell wall formation and growth in energy cane. Our study provides new insights on the regulation of the axillary bud of species of the Saccharum complex.

Dwarf and Increased Branching 1 controls plant height and axillary bud outgrowth in Medicago truncatula

Optimizing plant architecture is an efficient approach for breeders to increase crop yields, and phytohormones such as gibberellins (GAs) play an important role in controlling growth. Medicago truncatula is a model legume species, but the molecular mechanisms underlying its architecture are largely unknown. In this study, we examined a tobacco retrotransposon Tnt1-tagged mutant collection of M. truncatula and identified dwarf and increased branching 1 (dib1), which exhibited extreme dwarfism and increased numbers of lateral branches. By analysis of the flanking sequences of Tnt1 insertions in different alleles of the tagged lines, we were able to clone DIB1. Linkage analysis and reverse screening of the flanking-sequence tags identified Medtr2g102570 as the gene corresponding to the DIB1 locus in the dib1 loss-of-function mutants. Phylogenetic analysis indicated that DIB1 was the ortholog of PsGA3ox1/Le in Pisum sativum. Expression analysis using a GUS-staining reporter line showed that DIB1 was expressed in the root apex, pods, and immature seeds. Endogenous GA4 concentrations were markedly decreased whilst some of representative GA biosynthetic enzymes were up-regulated in the dib1 mutant. In addition, exogenous application of GA3 rescued the dib1 mutant phenotypes. Overall, our results suggest that DIB1 controls plant height and axillary bud outgrowth via an influence on the biosynthesis of bioactive GAs. DIB1 could therefore be a good candidate gene for breeders to optimize plant architecture for crop improvement.

Ascorbate glutathione-dependent H2O2 scavenging is an important process in axillary bud outgrowth in rosebush

Background and Aims Branching is an important mechanism of plant shape establishment and the direct consequence of axillary bud outgrowth. Recently, hydrogen peroxide (H2O2) metabolism, known to be involved in plant growth and development, has been proposed to contribute to axillary bud outgrowth. However, the involvement of H2O2 in this process remains unclear. Methods We analysed the content of H2O2 during bud outgrowth and characterized its catabolism, both at the transcriptional level and in terms of its enzymatic activities, using RT–qPCR and spectrophotometric methods, respectively. In addition, we used in vitro culture to characterize the effects of H2O2 application and the reduced glutathione (GSH) synthesis inhibitor l-buthionine sulfoximine (BSO) on bud outgrowth in relation to known molecular markers involved in this process. Key Results Quiescent buds displayed a high content of H2O2 that declined when bud outgrowth was initiated, as the consequence of an increase in the scavenging activity that is associated with glutathione pathways (ascorbate–glutathione cycle and glutathione biosynthesis); catalase did not appear to be implicated. Modification of bud redox state after the application of H2O2 or BSO prevented axillary bud outgrowth by repressing organogenesis and newly formed axis elongation. Hydrogen peroxide also repressed bud outgrowth-associated marker gene expression. Conclusions These results show that high levels of H2O2 in buds that are in a quiescent state prevents bud outgrowth. Induction of ascorbate–glutathione pathway scavenging activities results in a strong decrease in H2O2 content in buds, which finally allows bud outgrowth.

The role of trehalose 6-phosphate in shoot branching – local and non-local effects on axillary bud outgrowth in arabidopsis rosettes

SUMMARY- Trehalose 6-phosphate (Tre6P) is a sucrose signalling metabolite that has been implicated in regulation of shoot branching, but its precise role is not understood.- We expressed tagged forms of TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1) to determine where Tre6P is synthesized in arabidopsis (Arabidopsis thaliana), and investigated the impact of localized changes in Tre6P levels, in axillary buds or vascular tissues, on shoot branching in wild-type and branching mutant backgrounds.- TPS1 is expressed in axillary buds and the subtending vasculature, as well as in the leaf and stem vasculature. Expression of a heterologous trehalose-6-phosphate phosphatase (TPP) to lower Tre6P in axillary buds strongly delayed bud outgrowth in long days and inhibited branching in short days. TPP expression in the vasculature also delayed lateral bud outgrowth and decreased branching. Increased Tre6P in the vasculature enhanced branching and was accompanied by higher expression of FLOWERING LOCUS T (FT) and up-regulation of sucrose transporters. Increased vascular Tre6P levels enhanced branching in branched1 but not in ft mutant backgrounds.- These results provide direct genetic evidence of a local role for Tre6P in regulation of axillary bud outgrowth within the buds themselves, and also connect Tre6P with systemic regulation of shoot branching via FT.

Transcriptomic and physiological analyses of rice seedlings under different nitrogen supplies provide insight into the regulation involved in axillary bud outgrowth

Background: N is an important macronutrient required for plant development and significantly influences axillary bud outgrowth, which affects tillering and grain yields of rice. However, how different N concentrations affect axillary bud growth at the molecular and transcriptional levels remains unclear. Results: In this study, morphological changes in the axillary bud growth of rice seedlings under different N concentrations ranging from low to high levels were systematically observed. To investigate the expression of N-induced genes involved in axillary bud growth, we used RNA-seq technology to generate mRNA transcriptomic data from two tissue types, basal parts and axillary buds, of plants grown under six different N concentrations. In total, 10,221 and 12,180 DEGs induced by LN or HN supplies were identified in the basal parts and axillary buds, respectively, via comparisons to expression levels under NN level. Analysis of the coexpression modules from the DEGs of the basal parts and axillary buds revealed an abundance of related biological processes underlying the axillary bud growth of plants under N treatments. Among these processes, the activity of cell division and expansion was positively correlated with the growth rate of axillary buds of plants grown under different N supplies. Additionally, TFs and phytohormones were shown to play crucial roles in determining the axillary bud growth of plants grown under different N concentrations. Further validation of OsGS1;2 and OsGS2 , the rice mutants of which presented altered tiller numbers, validated our transcriptomic data. Conclusion: These results indicate that different N concentrations affect the axillary bud growth rate, and our study revealed comprehensive expression profiles of genes that respond to different N concentrations, providing an important resource for future studies attempting to determine how axillary bud growth is controlled by different N supplies.

Transcriptomic and physiological analyses of rice seedlings under different nitrogen supplies provide insight into the regulation involved in axillary bud outgrowth

Background: N is an important macronutrient required for plant development and significantly influences axillary bud outgrowth, which affects tillering and grain yields of rice. However, how different N concentrations affect axillary bud growth at the molecular and transcriptional levels remains unclear. Results: In this study, morphological changes in the axillary bud growth of rice seedlings under different N concentrations ranging from low to high levels were systematically observed. To investigate the expression of N-induced genes involved in axillary bud growth, we used RNA-seq technology to generate mRNA transcriptomic data from two tissue types, basal parts and axillary buds, of plants grown under six different N concentrations. In total, 10,221 and 12,180 DEGs induced by LN or HN supplies were identified in the basal parts and axillary buds, respectively, via comparisons to expression levels under NN level. Analysis of the coexpression modules from the DEGs of the basal parts and axillary buds revealed an abundance of related biological processes underlying the axillary bud growth of plants under N treatments. Among these processes, the activity of cell division and expansion was positively correlated with the growth rate of axillary buds of plants grown under different N supplies. Additionally, TFs and phytohormones were shown to play crucial roles in determining the axillary bud growth of plants grown under different N concentrations. Further validation of OsGS1;2 and OsGS2 , the rice mutants of which presented altered tiller numbers, validated our transcriptomic data. Conclusion: These results indicate that different N concentrations affect the axillary bud growth rate, and our study revealed comprehensive expression profiles of genes that respond to different N concentrations, providing an important resource for future studies attempting to determine how axillary bud growth is controlled by different N supplies.