Activation of apoplastic sugar at the transition stage may be essential for axillary bud outgrowth in the grasses.

Shoot branches develop from buds in leaf axils. Once formed from axillary meristems, the buds enter a transition stage before growing into branches. The buds may transition into dormancy if internal and environmental factors limit sucrose supply to the buds. A fundamental question is why sucrose can be limiting at the transition stage for bud outgrowth, whereas new buds continue to be formed. Sucrose is transported to sink tissues through symplastic or apoplastic pathways and a shift from symplastic to apoplastic pathway is common during seed and fruit development. In addition, symplastic connected tissues are stronger sinks than symplastically isolated tissues that rely on sugars effluxed to the apoplast. Recent studies in sorghum, sugarcane, and maize indicate activation of apoplastic sugar in buds that transition to outgrowth but not to dormancy, although the mode of sugar transport during bud formation is still unclear. Since the apoplastic pathway in sorghum buds was specifically activated during bud outgrowth, we posit that sugar for axillary bud formation is most likely supplied through the symplastic pathway. This suggests a key developmental change at the transition stage, which alters the sugar transport pathway of newly-formed buds from symplastic to apoplastic, making the buds a less strong sink for sugars. We suggest therefore that bud outgrowth that relies on overflow of excess sucrose to the apoplast will be more sensitive to internal and environmental factors that enhance the growth of sink tissues and sucrose demand in the parent shoot; whereas bud formation that relies on symplastic sucrose will be less affected by these factors.

Identification of Phytotoxic Levels of Copper and Nickel in Commercial Organic Soil Amendments Recycled from Poultry Farms and Municipal Wastes.

Commercial-scale recycling of agricultural and municipal wastes into organic soil amendments facilitates safe disposal of waste and reduces environmental contamination. However, phytotoxicity of commercial organic amendments to crops is a major concern to farmers. Consistent with this, commercial chicken manure and Milorganite (recycled from municipal waste) were found to be phytotoxic. Chicken manure aqueous extract contains 10.8 ppm Cu and 0.7 ppm Ni. The level of Cu and Ni in Milorganite is lower. The current study identified an aqueous solution containing 5 ppm Cu, lower than in chicken manure aqueous extract, was highly phytotoxic to mustard seeds germination. Therefore, phytotoxicity of chicken manure is in part due to Cu. An aqueous solution containing 1 ppm Ni was not phytotoxic; whereas 0.125 ppm Ni was phytotoxic when 62.5 ppm Na, which is nontoxic, was added to the solution. Therefore, synergistic effects of chemicals in the organic amendments may induce phytotoxicity.

Shade signals alter the expression of circadian clock genes in newly-formed bioenergy sorghum internodes.

Stem internodes of bioenergy sorghum inbred R.07020 are longer at high plant density (shade) than at low plant density (control). Initially, the youngest newly-formed subapical stem internodes of shade-treated and control plants are comparable in length. However, full-length internodes of shade-treated plants are three times longer than the internodes of the control plants. To identify the early molecular events associated with internode elongation in response to shade, we analyzed the transcriptome of the newly-formed internodes of shade-treated and control plants sampled between 4 and 6 hr after the start of the light period (14 hr light/10 hr dark). Sorghum genes homologous to the Arabidopsis shade marker genes ATHB2 and PIL1 were not differentially expressed. The results indicate that shade signals promote internode elongation indirectly because sorghum internodes are not illuminated and grow while enclosed with leaf sheaths. Sorghum genes homologous to the Arabidopsis morning-phased circadian clock genes LHY, RVE, and LNK were downregulated and evening-phased genes such as TOC1, PRR5, and GI were upregulated in young internodes in response to shade. We hypothesize that a change in the function or patterns of expression of the circadian clock genes is the earliest molecular event associated with internode elongation in response to shade in bioenergy sorghum. Increased expression of CycD1, which promotes cell division, and decreased expression of cell wall-loosening and MBF1-like genes, which promote cell expansion, suggest that shade signals promote internode elongation in bioenergy sorghum in part through increasing cell number by delaying transition from cell division to cell expansion.

Evaluation of phytotoxicity of three organic amendments to collard greens using the seed germination bioassay.

Small-scale vegetable and fruit crop producers in the USA use locally available commercial organic fertilizers and soil amendments recycled from municipal and agricultural wastes. Organic soil amendments provide crops with their nutrient needs and maintain soil health by modifying its physical, chemical, and biological properties. However, organic soil amendments might add unwanted elements such as toxic heavy metals or salts, which might inhibit crop growth and reduce yield. Therefore, the objective of this study was to evaluate phytotoxicity of three commercial organic amendments, chicken manure, milorganite, and dairy manure, to collard greens using the seed germination bioassay and chemical analysis of the organic amendments. The seed germination bioassay was conducted by incubating collard greens seeds to germinate in 1:10 (w/v) organic amendment aqueous extracts. Results of this work identified phytotoxic effects of chicken manure and milorganite, but not dairy manure, to collard greens. Potentially phytotoxic chemicals such as copper, zinc, nickel, and salts were also higher in chicken manure and milorganite compared to dairy manure. In particular, nickel in chicken manure and milorganite aqueous extracts was 28-fold and 21-fold, respectively, higher than previously reported toxic levels to wheat seedlings. The results demonstrate the need for more research on phytotoxicity of commercial organic soil amendments to ensure their safe use in vegetable and fruit crop production systems.

A Growing Stem Inhibits Bud Outgrowth - The Overlooked Theory of Apical Dominance.

Three theories of apical dominance, direct, diversion, and indirect, were proposed in the 1930s to explain how auxin synthesized in the shoot apex might inhibit axillary bud outgrowth, and thus shoot branching. The direct and diversion theories of apical dominance have been investigated in detail, and they are replaced with the current auxin transport canalization and second messenger theories, respectively. These two current theories still cannot entirely explain the phenomenon of apical dominance. Although there is ample evidence that the inhibition of bud outgrowth by auxin from the shoot apex is linked to stem elongation and highly branched auxin biosynthesis or signaling mutants are dwarf, the third theory proposed in the 1930s, the indirect theory, that explains apical dominance as auxin-induced stem growth indirectly inhibits bud outgrowth has been overlooked. The indirect theory did not propose how a growing stem might inhibit bud outgrowth. Recent discoveries indicate bud dormancy (syn. quiescence, paradormancy) in response to intrinsic and environmental factors in diverse species is linked to enhanced growth of the main shoot and reduced sugar level in the buds. Since a growing stem is a strong sink for sugars, and sugar is indispensable for shoot branching, the indirect theory of apical dominance might now be explained as auxin-induced stem growth inhibits bud outgrowth by diverting sugars away from buds. Detailed study of the indirect theory and the effect of source-sink status on dormancy and outgrowth of axillary buds will advance our knowledge of apical dominance and shoot branching in plants.

Dynamics of gene expression during development and expansion of vegetative stem internodes of bioenergy sorghum.

BACKGROUND: Bioenergy sorghum accumulates 75% of shoot biomass in stem internodes. Grass stem internodes are formed during vegetative growth and elongate in response to developmental and environmental signals. To identify genes and molecular mechanisms that modulate the extent of internode growth, we conducted microscopic and transcriptomic analyses of four successive sub-apical vegetative internodes representing different stages of internode development of the bioenergy sorghum genotype R.07020. RESULTS: Stem internodes of sorghum genotype R.07020 are formed during the vegetative phase and their length is enhanced by environmental signals such as shade and floral induction in short days. During vegetative growth, the first visible and youngest sub-apical internode was ~0.7 cm in length, whereas the fourth fully expanded internode was ~5 cm in length. Microscopic analyses revealed that all internode tissue types including pith parenchyma and vascular bundles are present in the four successive internodes. Growth in the first two sub-apical internodes occurred primarily through an increase in cell number consistent with expression of genes involved in the cell cycle and DNA replication. Growth of the 3rd internode was associated with an increase in cell length and growth cessation in the 4th internode was associated with up-regulation of genes involved in secondary cell wall deposition. The expression of genes involved in hormone metabolism and signaling indicates that GA, BR, and CK activity decreased while ethylene, ABA, and JA increased in the 3rd/4th internodes. While the level of auxin appears to be increasing as indicated by the up-regulation of ARFs, down-regulation of TIR during development indicates that auxin signaling is also modified. The expression patterns of transcription factors are closely associated with their role during the development of the vegetative internodes. CONCLUSIONS: Microscopic and transcriptome analyses of four successive sub-apical internodes characterized the developmental progression of vegetative stem internodes from initiation through full elongation in the sorghum genotype R.07020. Transcriptome profiling indicates that dynamic variation in the levels and action of GA, CK, IAA, BR, ethylene, ABA, and JA modulate gene expression and growth during internode growth and development. This study provides detailed microscopic and transcriptomic data useful for identifying genes and molecular pathways regulating internode elongation in response to various developmental and environmental signals.

Transcriptome Profiling of Tiller Buds Provides New Insights into PhyB Regulation of Tillering and Indeterminate Growth in Sorghum.

Phytochrome B (phyB) enables plants to modify shoot branching or tillering in response to varying light intensities and ratios of red and far-red light caused by shading and neighbor proximity. Tillering is inhibited in sorghum genotypes that lack phytochrome B (58M, phyB-1) until after floral initiation. The growth of tiller buds in the first leaf axil of wild-type (100M, PHYB) and phyB-1 sorghum genotypes is similar until 6 d after planting when buds of phyB-1 arrest growth, while wild-type buds continue growing and develop into tillers. Transcriptome analysis at this early stage of bud development identified numerous genes that were up to 50-fold differentially expressed in wild-type/phyB-1 buds. Up-regulation of terminal flower1, GA2oxidase, and TPPI could protect axillary meristems in phyB-1 from precocious floral induction and decrease bud sensitivity to sugar signals. After bud growth arrest in phyB-1, expression of dormancy-associated genes such as DRM1, GT1, AF1, and CKX1 increased and ENOD93, ACCoxidase, ARR3/6/9, CGA1, and SHY2 decreased. Continued bud outgrowth in wild-type was correlated with increased expression of genes encoding a SWEET transporter and cell wall invertases. The SWEET transporter may facilitate Suc unloading from the phloem to the apoplast where cell wall invertases generate monosaccharides for uptake and utilization to sustain bud outgrowth. Elevated expression of these genes was correlated with higher levels of cytokinin/sugar signaling in growing buds of wild-type plants.

Tillering in the sugary1 sweet corn is maintained by overriding the teosinte branched1 repressive signal.

The evolution of apical dominance in maize during domestication from teosinte is associated with higher expression from the teosinte branched1 (tb1) gene that inhibits tiller bud outgrowth. Unlike many standard maize varieties, the sweet corn inbred P39 that carries a mutation in a starch biosynthesis gene sugary1 produces multiple tillers and providing an opportunity to explore the diversification of the tb1 signal in maize. Through gene expression analysis, we show that tiller buds in P39 continue to grow by overriding the high expression level of tb1 that arrests bud outgrowth in maize inbred B73. In addition, we demonstrate that while B73 is largely non-responsive to shade, both P39 and teosinte respond through tb1-independent and tb1-dependent molecular mechanisms, respectively, leading to inhibition of tiller bud outgrowth.

Photosynthetic leaf area modulates tiller bud outgrowth in sorghum.

Shoot branches or tillers develop from axillary buds. The dormancy versus outgrowth fates of buds depends on genetic, environmental and hormonal signals. Defoliation inhibits bud outgrowth indicating the role of leaf-derived metabolic factors such as sucrose in bud outgrowth. In this study, the sensitivity of bud outgrowth to selective defoliation was investigated. At 6 d after planting (6 DAP), the first two leaves of sorghum were fully expanded and the third was partially emerged. Therefore, the leaves were selectively defoliated at 6 DAP and the length of the bud in the first leaf axil was measured at 8 DAP. Bud outgrowth was inhibited by defoliation of only 2 cm from the tip of the second leaf blade. The expression of dormancy and sucrose-starvation marker genes was up-regulated and cell cycle and sucrose-inducible genes was down-regulated during the first 24 h post-defoliation of the second leaf. At 48 h, the expression of these genes was similar to controls as the defoliated plant recovers. Our results demonstrate that small changes in photosynthetic leaf area affect the propensity of tiller buds for outgrowth. Therefore, variation in leaf area and photosynthetic activity should be included when integrating sucrose into models of shoot branching.

Grasses provide new insights into regulation of shoot branching.

Tillering (branching) is a major determinant of crop yield that is controlled by complex interactions between hormonal, developmental, and environmental factors. Historically, research on shoot branching has focused on eudicots, mainly due to the ease of manipulating branching by shoot decapitation and grafting in these species. These studies demonstrated hormonal control of branching. Recent studies in monocots have contributed to our knowledge of tillering/branching by identifying novel branching genes and regulatory mechanisms. A comparison of branching controls in eudicots and monocots reveals that the regulatory signals and genes are broadly conserved, but that there are differences in the detail.

Physiological perspectives of reduced tillering and stunting in the tiller inhibition (tin) mutant of wheat.

The number of tillers established in cereal crops far exceeds the number that end up being grain bearing at maturity. Improving the economy in tillering has been proposed to improve cereal yields in both favourable and unfavourable environments. The tiller inhibition mutant (tin) is potentially useful for breeding varieties with a greater economy of tillering. However, its tendency to stunting under long day and low temperatures has limited its use. Recently, the inhibition of tillering in tin has been linked to precocious development of solid basal internodes that compete for sucrose and possibly other resources with the growing tiller buds leading to their developmental arrest. Although the physiological basis of stunting in tin is unknown, both inhibition of tillering and stunting begin during the transition from vegetative to reproductive phase indicating a common physiological basis for both. In this review, we provide overall perspectives for the physiological basis of tiller inhibition and stunting in tin and suggest the direction of research in the future.

Inhibition of tiller bud outgrowth in the tin mutant of wheat is associated with precocious internode development.

Tillering (branching) is a major yield component and, therefore, a target for improving the yield of crops. However, tillering is regulated by complex interactions of endogenous and environmental signals, and the knowledge required to achieve optimal tiller number through genetic and agronomic means is still lacking. Regulatory mechanisms may be revealed through physiological and molecular characterization of naturally occurring and induced tillering mutants in the major crops. Here we characterize a reduced tillering (tin, for tiller inhibition) mutant of wheat (Triticum aestivum). The reduced tillering in tin is due to early cessation of tiller bud outgrowth during the transition of the shoot apex from the vegetative to the reproductive stage. There was no observed difference in the development of the main stem shoot apex between tin and the wild type. However, tin initiated internode development earlier and, unlike the wild type, the basal internodes in tin were solid rather than hollow. We hypothesize that tin represents a novel type of reduced tillering mutant associated with precocious internode elongation that diverts sucrose (Suc) away from developing tillers. Consistent with this hypothesis, we have observed upregulation of a gene induced by Suc starvation, downregulation of a Suc-inducible gene, and a reduced Suc content in dormant tin buds. The increased expression of the wheat Dormancy-associated (DRM1-like) and Teosinte Branched1 (TB1-like) genes and the reduced expression of cell cycle genes also indicate bud dormancy in tin. These results highlight the significance of Suc in shoot branching and the possibility of optimizing tillering by manipulating the timing of internode elongation.

grassy tillers1 promotes apical dominance in maize and responds to shade signals in the grasses.

The shape of a plant is largely determined by regulation of lateral branching. Branching architecture can vary widely in response to both genotype and environment, suggesting regulation by a complex interaction of autonomous genetic factors and external signals. Tillers, branches initiated at the base of grass plants, are suppressed in response to shade conditions. This suppression of tiller and lateral branch growth is an important trait selected by early agriculturalists during maize domestication and crop improvement. To understand how plants integrate external environmental cues with endogenous signals to control their architecture, we have begun a functional characterization of the maize mutant grassy tillers1 (gt1). We isolated the gt1 gene using positional cloning and found that it encodes a class I homeodomain leucine zipper gene that promotes lateral bud dormancy and suppresses elongation of lateral ear branches. The gt1 expression is induced by shading and is dependent on the activity of teosinte branched1 (tb1), a major domestication locus controlling tillering and lateral branching. Interestingly, like tb1, gt1 maps to a quantitative trait locus that regulates tillering and lateral branching in maize and shows evidence of selection during maize domestication. Branching and shade avoidance are both of critical agronomic importance, but little is known about how these processes are integrated. Our results indicate that gt1 mediates the reduced branching associated with the shade avoidance response in the grasses. Furthermore, selection at the gt1 locus suggests that it was involved in improving plant architecture during the domestication of maize.

The developmental dynamics of the maize leaf transcriptome.

We have analyzed the maize leaf transcriptome using Illumina sequencing. We mapped more than 120 million reads to define gene structure and alternative splicing events and to quantify transcript abundance along a leaf developmental gradient and in mature bundle sheath and mesophyll cells. We detected differential mRNA processing events for most maize genes. We found that 64% and 21% of genes were differentially expressed along the developmental gradient and between bundle sheath and mesophyll cells, respectively. We implemented Gbrowse, an electronic fluorescent pictograph browser, and created a two-cell biochemical pathway viewer to visualize datasets. Cluster analysis of the data revealed a dynamic transcriptome, with transcripts for primary cell wall and basic cellular metabolism at the leaf base transitioning to transcripts for secondary cell wall biosynthesis and C(4) photosynthetic development toward the tip. This dataset will serve as the foundation for a systems biology approach to the understanding of photosynthetic development.

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 paradormancy. 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 out-growth 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.

Phytochrome regulation of branching in Arabidopsis.

The red light:far-red light ratio perceived by phytochromes controls plastic traits of plant architecture, including branching. Despite the significance of branching for plant fitness and productivity, there is little quantitative and mechanistic information concerning phytochrome control of branching responses in Arabidopsis (Arabidopsis thaliana). Here, we show that in Arabidopsis, the negative effects of the phytochrome B mutation and of low red light:far-red light ratio on branching were largely due to reduced bud outgrowth capacity and an increased degree of correlative inhibition acting on the buds rather than due to a reduced number of leaves and buds available for branching. Phytochrome effects on the degree of correlative inhibition required functional BRANCHED1 (BRC1), BRC2, AXR1, MORE AXILLARY GROWTH2 (MAX2), and MAX4. The analysis of gene expression in selected buds indicated that BRC1 and BRC2 are part of different gene networks. The BRC1 network is linked to the growth capacity of specific buds, while the BRC2 network is associated with coordination of growth among branches. We conclude that the branching integrators BRC1 and BRC2 are necessary for responses to phytochrome, but they contribute differentially to these responses, likely acting through divergent pathways.

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.

The molecular analysis of the shade avoidance syndrome in the grasses has begun.

The shade avoidance syndrome (SAS) is a morphological and physiological response initiated by a decrease in light quantity and a change in light quality. Recent work in Arabidopsis thaliana has begun to define the molecular components of the SAS in a model dicot species, but little is known of these networks in agronomically important grasses. The focus of this review is to present a current view of the SAS in the grasses based largely on the characterization of mutants in the phytochrome signal transduction pathway and on the effects of far-red light treatments on plant growth. In cereal grasses, intense selection by plant breeders has acted to attenuate some but not all shade avoidance responses within modern crop varieties. Traditionally, breeding efforts have been focused on optimizing grain yield. However, with the recent interest in lignocellulosic-based biofuels, a new breeding paradigm may emerge to optimize biomass at the expense of grain yield. Some of the opportunities and challenges for engineering plant architecture to maximize resource use efficiency and yield by targeting the SAS in grasses are discussed.

Phytochrome B represses Teosinte Branched1 expression and induces sorghum axillary bud outgrowth in response to light signals.

Light is one of the environmental signals that regulate the development of shoot architecture. Molecular mechanisms regulating shoot branching by light signals have not been investigated in detail. Analyses of light signaling mutants defective in branching provide insight into the molecular events associated with the phenomenon. It is well documented that phytochrome B (phyB) mutant plants display constitutive shade avoidance responses, including increased plant height and enhanced apical dominance. We investigated the phyB-1 mutant sorghum (Sorghum bicolor) and analyzed the expression of the sorghum Teosinte Branched1 gene (SbTB1), which encodes a putative transcription factor that suppresses bud outgrowth, and the sorghum dormancy-associated gene (SbDRM1), a marker of bud dormancy. Buds are formed in the leaf axils of phyB-1; however, they enter into dormancy soon after their formation. The dormant state of phyB-1 buds is confirmed by the high level of expression of the SbDRM1 gene. The level of SbTB1 mRNA is higher in the buds of phyB-1 compared to wild type, suggesting that phyB mediates the growth of axillary shoots in response to light signals in part by regulating the mRNA abundance of SbTB1. These results are confirmed by growing wild-type seedlings with supplemental far-red light that induces shade avoidance responses. We hypothesize that active phyB (Pfr) suppresses the expression of the SbTB1 gene, thereby inducing bud outgrowth, whereas environmental conditions that inactivate phyB allow increased expression of SbTB1, thereby suppressing bud outgrowth.

Biomarker metabolites capturing the metabolite variance present in a rice plant developmental period.

BACKGROUND: This study analyzes metabolomic data from a rice tillering (branching) developmental profile to define a set of biomarker metabolites that reliably captures the metabolite variance of this plant developmental event, and which has potential as a basis for rapid comparative screening of metabolite profiles in relation to change in development, environment, or genotype. Changes in metabolism, and in metabolite profile, occur as a part of, and in response to, developmental events. These changes are influenced by the developmental program, as well as external factors impinging on it. Many samples are needed, however, to characterize quantitative aspects of developmental variation. A biomarker metabolite set could benefit screening of quantitative plant developmental variation by providing some of the advantages of both comprehensive metabolomic studies and focused studies of particular metabolites or pathways. RESULTS: An appropriate set of biomarker metabolites to represent the plant developmental period including the initiation and early growth of rice tillering (branching) was obtained by: (1) determining principal components of the comprehensive metabolomic profile, then (2) identifying clusters of metabolites representing variation in loading on the first three principal components, and finally (3) selecting individual metabolites from these clusters that were known to be common among diverse organisms. The resultant set of 21 biomarker metabolites was reliable (P = 0.001) in capturing 83% of the metabolite variation in development. Furthermore, a subset of the biomarker metabolites was successful (P = 0.05) in correctly predicting metabolite change in response to environment as determined in another rice metabolomics study. CONCLUSION: The ability to define a set of biomarker metabolites that reliably captures the metabolite variance of a plant developmental event was established. The biomarker metabolites are all commonly present in diverse organisms, so studies of their quantitative relationships can provide comparative information concerning metabolite profiles in relation to change in plant development, environment, or genotype.