Non‐dormant Axillary Bud 1 regulates axillary bud outgrowth in sorghum

Tillering contributes to grain yield and plant architecture and therefore is an agronomically important trait in sorghum (Sorghum bicolor). Here, we identified and functionally characterized a mutant of the Non‐dormant Axillary Bud 1 (NAB1) gene from an ethyl methanesulfonate‐mutagenized sorghum population. The nab1 mutants have increased tillering and reduced plant height. Map‐based cloning revealed that NAB1 encodes a carotenoid‐cleavage dioxygenase 7 (CCD7) orthologous to rice (Oryza sativa) HIGH‐TILLERING DWARF1/DWARF17 and Arabidopsis thaliana MORE AXILLARY BRANCHING 3. NAB1 is primarily expressed in axillary nodes and tiller bases and NAB1 localizes to chloroplasts. The nab1 mutation causes outgrowth of basal axillary buds; removing these non‐dormant basal axillary buds restored the wild‐type phenotype. The tillering of nab1 plants was completely suppressed by exogenous application of the synthetic strigolactone analog GR24. Moreover, the nab1 plants had no detectable strigolactones and displayed stronger polar auxin transport than wild‐type plants. Finally, RNA‐seq showed that the expression of genes involved in multiple processes, including auxin‐related genes, was significantly altered in nab1. These results suggest that NAB1 functions in strigolactone biosynthesis and the regulation of shoot branching via an interaction with auxin transport.     

Suppression of sorghum axillary bud outgrowth by shade, phyB and defoliation signalling pathways

In recent years, several genetic components of vegetative axillary bud development have been defined in both monocots and eudicots, but our understanding of environmental inputs on branching remains limited. Recent work in sorghum ( Sorghum bicolor ) has revealed a role for phytochrome B (phyB) in the control of axillary bud outgrowth through the regulation of Teosinte Branched1 ( TB1 ) gene. In maize ( Zea mays ), TB1 is a dosage-dependent inhibitor of axillary meristem progression, and the expression level of TB1 is a sensitive measure of axillary branch development. To further explore the mechanistic basis of branching, the expression of branching and cell cycle-related genes were examined in phyB-1 and wild-type sorghum axillary buds following treatment with low-red : far-red light and defoliation. Although defoliation inhibited bud outgrowth, it did not influence the expression of sorghum TB1 ( SbTB1 ), whereas changes in SbMAX2 expression, a homolog of the Arabidopsis ( Arabidopsis thaliana ) branching inhibitor MAX2 , were associated with the regulation of bud outgrowth by both light and defoliation. The expression of several cell cycle-related genes was also decreased dramatically in buds repressed by defoliation, but not by phyB deficiency. The data suggest that there are at least two distinct molecular pathways that respond to light and endogenous signals to regulate axillary bud outgrowth.     

Phytochrome B and shade signals regulate phytochrome A expression

The control of phytochrome A expression at the protein and mRNA levels was investigated in wild-type and phyB-1 mutant sorghum ( Sorghum bicolor [L.] Moench). PHYA mRNA abundance follows a diurnal rhythm in both genotypes, with maximal accumulation near the latter part of the light period. PHYA mRNA is more abundant in the phyB-1 mutant. The level of PHYA message correlates with both R : FR and photon flux density in wild-type, but only with photon flux density in the phyB-1 mutant. The differences in mRNA abundance are reflected in the level of phyA protein, which is elevated in the phyB-1 mutant and accumulates under low photon flux density. During de-etiolation, PHYA message accumulation is initially repressed solely by a very low fluence response (VLFR) presumably mediated by phyA. The phyB-mediated low fluence response maintains the repression of accumulation past the time controlled by the VLFR. With repetitive photoperiods, the transition from the etiolated growth form to autotrophic competency is accompanied by a transition from light-induced reduction of PHYA mRNA abundance to enhanced accumulation during the light period. The loss of phyB function allows partial de-repression of PHYA message accumulation under repetitive photoperiods, resulting in plants deficient in phyB but enriched in phyA. The modification of PHYA mRNA and protein levels in the phyB-1 mutant documented in this study may help clarify the molecular basis of the phyB-1 phenotype. The tailoring of phyA abundance in wild-type to the time of day and shade signals suggests a plastic role for this pigment in controlling development in light-grown plants.     

Branching gene expression during chrysanthemum axillary bud outgrowth regulated by strigolactone and auxin transport

Shoot branching is essential in ornamental chrysanthemum production and determines final plant shape and quality. Auxin is associated with apical dominance to indirectly inhibit bud outgrowth. Two non-mutually exclusive models exist for indirect auxin inhibition. Basipetal auxin transport inhibits axillary bud outgrowth by limiting auxin export from buds to stem (canalization model) or by increasing strigolactone levels (second messenger model). Here we analyzed bud outgrowth in treatments with auxin (IAA), strigolactone (GR24) and auxin transport inhibitor (NPA) using a split-plate bioassay with isolated chrysanthemum stem segments. Besides measuring bud length, dividing cell percentage was measured with flow cytometry and RT-qPCR was used to monitor expression levels of genes involved in auxin transport (CmPIN1) and signaling (CmAXR2), bud dormancy (CmBRC1, CmDRM1) and strigolactone biosynthesis (CmMAX1, CmMAX3). Treatments over a 5-day period showed bud outgrowth in the control and inhibition with IAA and IAA?+?GR24. Bud outgrowth in the control coincided with high dividing cell percentage, decreased expression of CmBRC1 and CmDRM1 and increased CmPIN1 expression. Inhibition by IAA and IAA?+?GR24 coincided with low dividing cell percentage and unchanged or increased expressions of CmBRC1, CmDRM1 and CmPIN1. Treatment with GR24 showed restricted bud outgrowth that was counteracted by NPA. This restricted bud outgrowth was still concomitant with a high dividing cell percentage and coincided with decreased expression of dormancy genes. These results indicate incomplete inhibition of bud outgrowth by GR24 treatment and suggest involvement of auxin transport in the mechanism of bud inhibition by strigolactones, supporting the canalization model.     

Vegetative axillary bud dormancy induced by shade and defoliation signals in the grasses

Vegetative axillary bud dormancy and outgrowth is regulated by several hormonal and environmental signals. In perennials, the dormancy induced by hormonal and environmental signals has been categorized as eco-, endo- or para-dormancy. Over the past several decades para-dormancy has primarily been investigated in eudicot annuals. Recently, we initiated a study using the monoculm phyB mutant (phyB-1) and the freely branching near isogenic wild type (WT) sorghum (Sorghum bicolor) to identify molecular mechanisms and signaling pathways regulating dormancy and outgrowth of axillary buds in the grasses. In a paper published in the January 2010 issue of Plant Cell and Environment, we reported the role of branching genes in the inhibition of bud outgrowth by phyB, shade and defoliation signals. Here we present a model that depicts the molecular mechanisms and pathways regulating axillary bud dormancy induced by shade and defoliation signals in the grasses.Key words: axillary bud, dormancy, shade, phytochrome, defoliation, shoot branching, teosinte branched1, MAX2, cell cycle, sorghumThe dormancy and outgrowth of axillary buds is regulated by several plant hormones such as auxin, cytokinins, abscisic acid and strigolactones, and by environmental factors such as light quality, quantity and duration as well as water, temperature and nutrient status.^1–3 Since the fate of an axillary bud is regulated by such diverse hormonal and environmental signals and their interactions, the type of dormancy induced varies. In perennials, three types of bud dormancy have been identified.^4,5 Dormancy mediated by factors within the bud is known as endo-dormancy; while dormancy induced by factors within the plant but outside the bud is called paradormancy or correlative inhibition; the best known example being apical dominance. Dormancy induced due to unfavorable environmental conditions is known as eco-dormancy. Although there is an indepth knowledge about para-dormancy in annuals,⁶ few studies have been conducted on eco-dormancy. Similarly, studies of endo-dormancy have largely been restricted to low-temperature mediated growth-cessation of axillary buds of perennial plants.^7,8 To understand the regulation of dormancy and outgrowth of axillary buds in monocots, we initiated a study on the molecular mechanisms inhibiting bud outgrowth by shade and defoliation signals in sorghum. Our results published in the January 2010 issue of Plant, Cell & Environment indicate that different types of dormancy may be induced in axillary buds of annual grasses by various signals and there may be overlapping and independent molecular mechanisms mediating induction of axillary bud dormancy.     

Ureteric bud outgrowth in response to RET activation is mediated by phosphatidylinositol 3-kinase

The c-ret gene encodes a receptor tyrosine kinase (RET) essential for the development of the kidney and enteric nervous system. Activation of RET requires the secreted neurotrophin GDNF (glial cell line-derived neurotrophic factor) and its high affinity receptor, a glycosyl phosphatidylinositol-linked cell surface protein GFRalpha1. In the developing kidney, RET, GDNF, and GFRalpha1 are all required for directed outgrowth and branching morphogenesis of the ureteric bud epithelium. Using MDCK renal epithelial cells as a model system, activation of RET induces cell migration, scattering, and formation of filopodia and lamellipodia. RET-expressing MDCK cells are able to migrate toward a localized source of GDNF. In this report, the intracellular signaling mechanisms regulating RET-dependent migration and chemotaxis are examined. Activation of RET resulted in increased levels of phosphatidylinositol 3-kinase (PI3K) activity and Akt/PKB phosphorylation. This increase in PI3K activity is essential for regulating the GDNF response, since the specific inhibitor, LY294002, blocks migration and chemotaxis of MDCK cells. Using an in vitro organ culture assay, inhibition of PI3K completely blocks the GDNF-dependent outgrowth of ectopic ureter buds. PI3K is also essential for branching morphogenesis once the ureteric bud has invaded the kidney mesenchyme. The data suggest that activation of RET in the ureteric bud epithelium signals through PI3K to control outgrowth and branching morphogenesis.     

Effect of light quality (red: far-red ratio) and defoliation treatments applied at a single phytomer on axillary bud outgrowth in Trifolium repens L.

We studied the effects of light quality and defoliation on the rate of phytomer appearance and axillary bud outgrowth in white clover. The treatments were applied to one phytomer, a phytomer being defined as the structural unit comprising a node, internode, axillary bud, subtending leaf and two nodal root primordia. Light of a low red:far-red (R:FR) ratio (0.27) was applied to a target phytomer either (i) within the apical bud and then to the axillary bud after emergence of the phytomer from the apical bud, or (ii) to the axillary bud only after emergence. The light conditions were directed to these specific parts of the plant by collimating light from small FR light-emitting diodes; with this technique we were able to change the light quality without any change in the level of photosynthetically active radiation. The subtending leaf of the target phytomer was retained or defoliated when it had emerged from the apical bud. FR light applied from the time the phytomer was within the apical bud caused a delay in branch appearance at the target phytomer. In contrast, direct treatment of the axillary bud with FR light after it had emerged from the apical bud did not result in any delay in branch appearance. As the light treatment of the apical bud may have changed the light environment of any of the organs contained in the bud we were unable to ascribe the delay in branch appearance to light perception by any particular organ. However, indirect evidence leads to the conclusion that the likely site of light perception was the developing leaf subtending the axillary bud while it was the outermost phytomer within the apical bud. These results do not support the hypothesis that the R:FR ratio of light incident at an axillary bud site is the environmental factor that controls bud development. Defoliation of the unfolding leaf reduced the rate of phytomer appearance on the main stolon but had no immediate effect on branch appearance. As a consequence there was a reduction in the number of phytomers between the stolon apical meristem and the first phytomer with a branch. This is frequently taken to indicate a relaxation of apical dominance, but in this case was found not to involve a direct effect on bud activity. A current model of white clover growth suggests that there is integration of activity between apical meristems but independence of activity and response to the local micro-environment by axillary buds. In contrast, we found that (i) defoliation reduced phytomer appearance only at the main stolon apical meristem and not at all the meristems in the plant and (ii) that a change in the local light environment of an axillary bud had no discernible effect on bud activity once the bud had emerged from the apical bud but could delay branching if applied before emergence. These results are at variance with the predictions of the model.     

How to cluster gene expression dynamics in response to environmental signals

Organisms usually cope with change in the environment by altering the dynamic trajectory of gene expression to adjust the complement of active proteins. The identification of particular sets of genes whose expression is adaptive in response to environmental changes helps to understand the mechanistic base of gene-environment interactions essential for organismic development. We describe a computational framework for clustering the dynamics of gene expression in distinct environments through Gaussian mixture fitting to the expression data measured at a set of discrete time points. We outline a number of quantitative testable hypotheses about the patterns of dynamic gene expression in changing environments and gene-environment interactions causing developmental differentiation. The future directions of gene clustering in terms of incorporations of the latest biological discoveries and statistical innovations are discussed. We provide a set of computational tools that are applicable to modeling and analysis of dynamic gene expression data measured in multiple environments.     

phyB-1 sorghum maintains responsiveness to simulated shade, irradiance and red light: far-red light

The sorghum [Sorghum bicolor (L.) Moench] phyB-1 mutant exhibits a constitutive shade-avoidance phenotype including excessive shoot elongation. It was previously shown that this mutant also overproduces ethylene. Although phytochrome B (phyB) is assumed to be the pigment most important in sensing and transducing shade signals, the sorghum phyB-1 mutant still responds to light signals characteristic of shade. Specifically, it was determined that the leaf blade : leaf sheath elongation of phyB-1 is responsive to red : far red (R : FR), but this response is opposite that of wild type (WT). Reducing the photosynthetic photon flux density (PPFD) strongly reduced the leaf blade : leaf sheath of WT but did not affect phyB-1, demonstrating a role for phyB in sensing PPFD. Using light-emitting diode (LED) lighting, it was found that WT ethylene production was increased with low R : FR while PPFD had no effect. Conversely, phyB-1 ethylene production increased only with high PPFD, high R : FR which was the treatment resulting in the least ethylene production by WT. Elevated ethylene production inhibits shoot elongation, but may contribute to shade avoidance by reducing leaf blade : leaf sheath elongation. Ethylene responses to light treatments designed to promote or reduce phytochrome A (phyA) activity, and the analysis of PHYA levels in the two cultivars suggests that phyA could be involved in transducing shade signals in light-grown sorghum. Responses potentially tranduced by phyA are elevated in phyB-1 which also over-expresses PHYA.     

Maintenance of surrogate light chain expression induces developmental delay in early B cell compartment

The production of a mature B cell requires passage through a number of developmental checkpoints. The pre-BCR plays a critical role in passage through the pro-B cell/pre-B cell checkpoint, and thus plays a central role in regulating the differentiation of a B cell. Due to the significance of this receptor, it is imperative that pre-BCR expression and function are precisely regulated. In this study, we have investigated a system in which the regulation of the pre-BCR is altered. We have found that continued expression of components of the pre-BCR (lambda5) resulted in a delay in the kinetics of B cell maturation. Pro-B cells from normal mouse bone marrow retrovirally infected with lambda5 exhibited a delay in differentiation. As compared with wild-type cells at the same time point, there is a reduction in the presence of cell surface markers that indicate developmental progression, and there is a 6- to 16-fold decrease in the production of Ig-positive cells in B cell maturation assays. The capacity to alter B cell progression by modifying and extending pre-BCR expression argues that the receptor and its associated signals play a unique role in directing developmental outcomes.     

Twist2 contributes to termination of limb bud outgrowth and patterning through direct regulation of Grem1

Twist1 has been demonstrated to play critical roles in the early development of neural crest and mesodermally derived tissues including the limb. Twist2 has been less well characterised but its relatively late onset of expression suggests specific roles in the development of a number of organs. Expression of Twist2 within the developing limbs begins after formation of the limb bud and persists within the peripheral mesenchyme until digital rays condense. We have used RCAS-mediated overexpression in chick to investigate the function of Twist2 in limb development. Viral misexpression following injection into the lateral plate mesoderm results in a spectrum of hypoplastic limb phenotypes. These include generalized shortening of the entire limb, fusion of the autopod skeletal elements, loss of individual digits or distal truncation resulting in complete loss of the autopod. These phenotypes appear to result from a premature termination of limb outgrowth and manifest as defective growth in both the proximal-distal and anterior-posterior axes. In situ hybridisation analysis demonstrates that many components of the Shh/Grem1/Fgf regulatory loop that controls early limb growth and patterning are downregulated by Twist2 overexpression. Grem1 has a complementary expression pattern to Twist2 within the limb primordia and co-expression of both Grem1 and Twist2 results in a rescue of the Twist2 overexpression phenotype. We demonstrate that Twist proteins directly repress Grem1 expression via a regulatory element downstream of the open reading frame. These data indicate that Twist2 regulates early limb morphogenesis through a role in terminating the Shh/Grem1/Fgf autoregulatory loop.     

2,3,7,8-tetrachlorodibenzo-p-dioxin induces CYP1B1 expression in human luteinized granulosa cells

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a reproductive toxicant in multiple species; however, mechanisms and direct ovarian effects are poorly understood. DNA microarrays were used to characterize gene expression profiles of human luteinized granulosa cells (HLGCs) exposed to TCDD in primary cultures. Exposure to 10 nM TCDD for 24 h induced a significant increase in CYP1B1, while few other genes responded. TaqMan PCR and Western immunoblotting demonstrated that induction was dose-dependent. Additionally, the microsomal form of catechol-O-methyltransferase (COMT) was highly expressed in HLGCs, along with only fractional amounts of the soluble form. This is the first report of CYP1B1 and COMT expression, and CYP1B1 induction, in cells from the human ovary. The role of CYP1B1 in the oxidative metabolism of estrogens and potential generation of DNA adducts in the ovary may have significant consequences for oocyte quality, corpus luteum function, and ovarian carcinogenesis.