Cytokinin profiling in plant tissues using ultra-performance liquid chromatography–electrospray tandem mass spectrometry

Sugar has only recently been identified as a key player in triggering bud outgrowth, while hormonal control of bud outgrowth is already well established. To get a better understanding of sugar control, the present study investigated how sugar availability modulates the hormonal network during bud outgrowth in Rosa hybrida. Other plant models, for which mutants are available, were used when necessary. Buds were grown in vitro to manipulate available sugars. The temporal patterns of the hormonal regulatory network were assessed in parallel with bud outgrowth dynamics. Sucrose determined bud entrance into sustained growth in a concentration-dependent manner. Sustained growth was accompanied by sustained auxin production in buds, and sustained auxin export in a DR5::GUS-expressing pea line. Several events occurred ahead of sucrose-stimulated bud outgrowth. Sucrose upregulated early auxin synthesis genes (RhTAR1, RhYUC1) and the auxin efflux carrier gene RhPIN1, and promoted PIN1 abundance at the plasma membrane in a pPIN1::PIN1-GFP-expressing tomato line. Sucrose downregulated both RwMAX2, involved in the strigolactone-transduction pathway, and RhBRC1, a repressor of branching, at an early stage. The presence of sucrose also increased stem cytokinin content, but sucrose-promoted bud outgrowth was not related to that pathway. In these processes, several non-metabolizable sucrose analogues induced sustained bud outgrowth in R. hybrida, Pisum sativum, and Arabidopsis thaliana, suggesting that sucrose was involved in a signalling pathway. In conclusion, we identified potential hormonal candidates for bud outgrowth control by sugar. They are central to future investigations aimed at disentangling the processes that underlie regulation of bud outgrowth by sugar.

Sucrose is an early modulator of the key hormonal mechanisms controlling bud outgrowth in Rosa hybrida

Concerted control of irradiance, temperature, water availability, mineral nutrition and beneficial root-associated microorganisms significantly improves adventitious rooting in eucalypts and poplars, essentially by modulating auxin and carbohydrate metabolism.
 Eucalyptus and Populus are among the most economically relevant tree genera. Clonal propagation allows fast genetic gain obtained using elite genotypes. Adventitious rooting (AR), a complex and multifactorial process, is often the main limiting factor for tree cloning. Herein, practical and basic approaches to optimize clonal propagation of eucalypts and poplars, focusing on the main environmental control factors affecting it, are explored. Auxin homeostasis and function are central to AR. Irradiance quality and quantity, as well as temperature, can effectively modulate auxin availability, transport and activity. The interaction of carbohydrates, irradiance and temperature is also at the core of AR. Root architecture may be effectively modified by different N sources. Several macro and micronutrients impact on central factors of rhizogenesis, including energy metabolism, gene expression and enzymatic activities regulating auxin and other phytohormonal steady-states driving AR. Appropriate mineral nutrition is often determinant for successful AR and survival. Microbial associations with the root system and the rhizosphere, both bacterial and fungal, can have a role in auxin availability to cuttings, as well as improve disease resistance, nutrition and water relations. Significant cost reduction in clonal propagation systems of eucalypt and poplar are attainable with an adequate control of environmental factors, particularly for donor plants. Future studies should extend the molecular and physiological findings of basic research to the commercial propagation systems, and these, by their turn, should be explored to provide further advances in the basic understanding of this crucial developmental process for human economy.

Environmental control of adventitious rooting in Eucalyptus and Populus cuttings

In Pinus radiata D. Don, the transition from the juvenile to the mature phase is characterized by a reduction in the tree's organogenic potential, which is usually reverted in breeding programs by reinvigoration procedures to enable vegetative propagation. In this work, we have determined the best culture conditions for in vitro reinvigoration of radiata pine buds, tested different cytokinin (CK) types [N⁶-benzyladenine (BA), meta-topolin (mT) and trans-zeatin] and concentrations (25 and 50 µM), and studied the effect of culture conditions on endogenous CK and indole-3-acetic acid (IAA) levels at different stages of the organogenic process. To this end, the levels of 43 CKs and IAA were determined in P. radiata buds before and during the reinvigoration process. When BA or mT was applied to the induction medium, we did not observe any significant increase or decrease in endogenous isoprenoid CK content. We also report for the first time the presence of O-glucosides in non-treated P. radiata explants from the field and remark the importance of O-glucosides as storage forms.

Endogenous cytokinin and auxin profiles during in vitro organogenesis from vegetative buds of Pinus radiata adult trees

Light and temperature are two environmental factors that deeply affect bud outgrowth. However, little is known about their impact on the bud burst gradient along a stem and their interactions with the molecular mechanisms of bud burst control. We investigated this question in two acrotonic rose cultivars. We demonstrated that the darkening of distal buds or exposure to cold (5 °C) prior to transfer to mild temperatures (20 °C) both repress acrotony, allowing the burst of quiescent medial and proximal buds. We sequenced the strigolactone pathway MAX-homologous genes in rose and studied their expression in buds and internodes along the stem. Only expressions of RwMAX1, RwMAX2 and RwMAX4 were detected. Darkening of the distal part of the shoot triggered a strong increase of RwMAX2 expression in darkened buds and bark-phloem samples, whereas it suppressed the acropetal gradient of the expression of RwMAX1 observed in stems fully exposed to light. Cold treatment induced an acropetal gradient of expression of RwMAX1 in internodes and of RwMAX2 in buds along the stem. Our results suggest that the bud burst gradient along the stem cannot be explained by a gradient of expression of RwMAX genes but rather by their local level of expression at each individual position.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/pce.12191

Impacts of light and temperature on shoot branching gradient and expression of strigolactone synthesis and signalling genes in rose.

Involvement of cytokinins (CKs) in axillary bud growth of miniature rose was studied. Variation in root formation and axillary bud growth was induced by two indole 3-butyric acid (IBA) pretreatments in two cutting sizes. At six physiological developmental stages around the onset of axillary bud growth, concentrations of CKs were determined in both root and axillary bud tissue by liquid chromatography combined with electrospray tandem mass spectrometry (LC-ESP-MS/MS). Chronological early onset of axillary bud growth occurred in long cuttings pretreated at low IBA concentration, whereas physiological early root formation was associated with long cuttings and high IBA concentration. The CKs zeatin (Z), isopentenyl adenine (iP), zeatin riboside (ZR), dihydrozeatin riboside (DHZR), isopentenyl adenosine (iPA), zeatin O-glucoside (ZOG), zeatin riboside O-glucoside (ZROG), zeatin riboside 5′-monophosphate (ZRMP), and isopentenyl adenosine 5′-monophosphate (iPAMP) were detected. Concentrations of CKs in axillary bud tissue far exceeded those in root tissue. Indole 3-butyric acid pretreatment influenced the concentration of CKs in axillary bud tissue more than did cutting size, whereas pretreatments only slightly affected CKs in root tissue. The dominant CKs found were iPAMP and ZR. An early and large increase in iPAMP indicated rapid CK biosynthesis in rootless cuttings, suggesting that green parts, including the axillary bud, can synthesize CKs. At the onset of axillary bud growth an increase in concentration of Z, ZR, ZRMP, ZOG, and ZROG was largely coincident with a decrease in iPAMP, iPA, iP, and DHZR. After the onset of axillary bud growth, CK content largely decreased. These results strongly indicate a positive role for CKs in axillary bud growth, and presumably ZRMP, ZR, and Z are active in miniature rose.

Changes in Concentrations of Cytokinins (CKs) in Root and Axillary Bud Tissue of Miniature Rose Suggest that Local CK Biosynthesis and Zeatin-Type CKs Play Important Roles in Axillary Bud Growth

SummaryRooting and growth responses of miniature rose cuttings were investigated in an experiment in which four propagation temperatures, two photosynthetic photon flux densities (PPFDs) with five auxin (IBA) concentrations, cutting sizes and cutting positions, were combined factorially in a response-surface design. Most prominently, temperature, cutting size and auxin and their interactions, influenced root and shoot growth. A propagation temperature of 24.6°C, and IBA concentrations between 10–3 and 10–1M, depending on temperature, were optimal for root formation. Root formation in extra short cuttings was delayed at low IBA concentrations. Regarding root formation, IBA could substitute for increased temperature as well as for increased cutting size. Onset of axillary bud growth was fastest at 24.6°C, and delayed in extra short cuttings. Application of IBA at 10–4 to 10–3M was optimal for axillary bud growth. By increasing the IBA concentration both time to flowering and plant height increased at 24.6°C...

Propagation temperature, PPFD, auxin treatment, cutting size and cutting position affect root formation, axillary bud growth and shoot development in miniature rose (Rosa hybrida L.) plants and alter homogeneity

SummaryThe effects of propagation soil temperature and photosynthetic photon flux density (PPFD), indole-3-butyric acid (IBA) concentration, and cutting position on root formation, axillary bud growth, and further growth were investigated in Schlumbergera ‘Russian Dancer’. Temperatures between 21.3oC and 24.7oC were optimal for root formation, whereas increasing temperature promoted axillary bud growth (onset of new phylloclade growth). At temperatures below 24.7oC, root formation and axillary bud growth occurred more or less simultaneously, whereas at higher temperatures axillary bud growth occurred before roots were visible. Temperature also affected the number of phylloclades on the second and third levels of stem segments produced after propagation. On increasing PPFD from 46 to 72 µmol m–2 s–1 during propagation, time to axillary bud growth was reduced by 15%, and more phylloclades were produced at high PPFD combined with high temperature. Rooting ability and axillary bud growth were affected by cutt...

Influence of propagation temperature, photosynthetic photon flux density, auxin treatment and cutting position on root formation, axillary bud growth and shoot development in Schlumbergera ‘Russian Dancer’

Niels Bent Bredmose

Papers

Changes in Concentrations of Cytokinins (CKs) in Root and Axillary Bud Tissue of Miniature Rose Suggest that Local CK Biosynthesis and Zeatin-Type CKs Play Important Roles in Axillary Bud Growth

Propagation temperature, PPFD, auxin treatment, cutting size and cutting position affect root formation, axillary bud growth and shoot development in miniature rose (Rosa hybrida L.) plants and alter homogeneity

Influence of propagation temperature, photosynthetic photon flux density, auxin treatment and cutting position on root formation, axillary bud growth and shoot development in Schlumbergera ‘Russian Dancer’