Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity

Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the cytokinin oxidase/dehydrogenase (AtCKX) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased cytokinin breakdown (30 to 45% of wild-type cytokinin content) and reduced expression of the cytokinin reporter gene ARR5:GUS (beta-glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in cytokinin biology. Analyses of promoter:GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of cytokinin functions.     

Root system architecture in Arabidopsis grown in culture is regulated by sucrose uptake in the aerial tissues

This article presents a detailed model for the regulation of lateral root formation in Arabidopsis thaliana seedlings grown in culture. We demonstrate that direct contact between the aerial tissues and sucrose in the growth media is necessary and sufficient to promote emergence of lateral root primordia from the parent root. Mild osmotic stress is perceived by the root, which then sends an abscisic acid-dependent signal that causes a decrease in the permeability of aerial tissues; this reduces uptake of sucrose from the culture media, which leads to a repression of lateral root formation. Osmotic repression of lateral root formation in culture can be overcome by mutations that cause the cuticle of a plant's aerial tissues to become more permeable. Indeed, we report here that the previously described lateral root development2 mutant overcomes osmotic repression of lateral root formation because of a point mutation in Long Chain Acyl-CoA Synthetase2, a gene essential for cutin biosynthesis. Together, our findings (1) impact the interpretation of experiments that use Arabidopsis grown in culture to study root system architecture; (2) identify sucrose as an unexpected regulator of lateral root formation; (3) demonstrate mechanisms by which roots communicate information to aerial tissues and receive information in turn; and (4) provide insights into the regulatory pathways that allow plants to be developmentally plastic while preserving the essential balance between aboveground and belowground organs.     

Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation

Ethylene represents an important regulatory signal for root development. Genetic studies in Arabidopsis thaliana have demonstrated that ethylene inhibition of root growth involves another hormone signal, auxin. This study investigated why auxin was required by ethylene to regulate root growth. We initially observed that ethylene positively controls auxin biosynthesis in the root apex. We subsequently demonstrated that ethylene-regulated root growth is dependent on (1) the transport of auxin from the root apex via the lateral root cap and (2) auxin responses occurring in multiple elongation zone tissues. Detailed growth studies revealed that the ability of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid to inhibit root cell elongation was significantly enhanced in the presence of auxin. We conclude that by upregulating auxin biosynthesis, ethylene facilitates its ability to inhibit root cell expansion.     

Auxin and ethylene: collaborators or competitors?

The individual roles of auxin and ethylene in controlling the growth and development of young seedlings have been well studied. In recent years, these two hormones have been shown to act synergistically to control specific growth and developmental processes, such as root elongation and root hair formation, as well as antagonistically in other processes, such as lateral root formation and hypocotyl elongation. This review examines the growth and developmental processes that are regulated by crosstalk between these two hormones and explores the mechanistic basis for the regulation of these processes. The emerging trend from these experiments is that ethylene modulates auxin synthesis, transport, and signaling with unique targets and responses in a range of tissues to fine-tune seedling growth and development.     

Transgenic analysis of sugar beet xyloglucan endo-transglucosylase/hydrolase Bv-XTH1 and Bv-XTH2 promoters reveals overlapping tissue-specific and wound-inducible expression profiles

The identification and analysis of tissue-specific gene regulatory elements will improve our knowledge of the molecular mechanisms that control the growth and development of different plant tissues and offer potentially useful tools for the genetic engineering of plants. A polymerase chain reaction (PCR)-based 5'-genome walk from sequences of an isolated sugar beet xyloglucan endo-transglucosylase hydrolase (XTH) gene led to the isolation of two independent upstream fragments. They were 1332 and 2163 base pairs upstream of the XTH ATG start site, respectively. In vivo transgenic assays in sugar beet hairy roots and Arabidopsis thaliana revealed that both fragments had promoter function and, in A. thaliana, directed expression in vascular tissues within the root, leaves and petals. Promoter activity was also observed in the leaf trichomes and within rapidly expanding stem internodes. Expression driven by both promoters was found to be wound inducible. Overall, the spatial and temporal expression pattern of these promoters suggested that the corresponding Bv-XTH genes (designated Bv-XTH1 and Bv-XTH2) may be involved in secondary cell wall formation. This work provides new insights on molecular mechanisms that could be exploited for the genetic engineering of sugar beet crops.     

Short-Root regulates primary, lateral, and adventitious root development in Arabidopsis

Short-Root (SHR) is a well-characterized regulator of radial patterning and indeterminacy of the Arabidopsis (Arabidopsis thaliana) primary root. However, its role during the elaboration of root system architecture remains unclear. We report that the indeterminate wild-type Arabidopsis root system was transformed into a determinate root system in the shr mutant when growing in soil or agar. The root growth behavior of the shr mutant results from its primary root apical meristem failing to initiate cell division following germination. The inability of shr to reactivate mitotic activity in the root apical meristem is associated with the progressive reduction in the abundance of auxin efflux carriers, PIN-FORMED1 (PIN1), PIN2, PIN3, PIN4, and PIN7. The loss of primary root growth in shr is compensated by the activation of anchor root primordia, whose tissues are radially patterned like the wild type. However, SHR function is not restricted to the primary root but is also required for the initiation and patterning of lateral root primordia. In addition, SHR is necessary to maintain the indeterminate growth of lateral and anchor roots. We conclude that SHR regulates a wide array of Arabidopsis root-related developmental processes.     

Ammonium inhibition of Arabidopsis root growth can be reversed by potassium and by auxin resistance mutations aux1, axr1, and axr2.

A novel effect of ammonium ions on root growth was investigated to understand how environmental signals affect organ development. Ammonium ions (3-12 mM) were found to dramatically inhibit Arabidopsis thaliana seedling root growth in the absence of potassium even if nitrate was present. This inhibition could be reversed by including in the growth medium low levels (20-100 microM) of potassium or alkali ions Rb+ and Cs+ but not alkali ions Na+ and Li+. The protective effect of low concentrations of potassium is not due to an inhibition of ammonium uptake. Ammonium inhibition is reversible, because root growth was restored in ammonium-treated seedlings if they were subsequently transferred to medium containing potassium. It is known that plant hormones can inhibit root growth. We found that mutants of Arabidopsis resistant to high levels of auxin and other hormones (aux1, axr1, and axr2) are also resistant to the ammonium inhibition and produce roots in the absence of potassium. Thus, the mechanisms that mediate the ammonium inhibition of root development are linked to hormone metabolic or signaling pathways. These findings have important implications for understanding how environmental signals, especially mineral nutrients, affect plant root development.     

Iso-anchorene is an endogenous metabolite that inhibits primary root growth in Arabidopsis

Carotenoid-derived regulatory metabolites and hormones are generally known to arise through the oxidative cleavage of a single double bond in the carotenoid backbone, which yields mono-carbonyl products called apocarotenoids. However, the extended conjugated double bond system of these pigments predestines them also to repeated cleavage forming dialdehyde products, diapocarotenoids, which have been less investigated due to their instability and low abundance. Recently, we reported on the short diapocarotenoid anchorene as an endogenous Arabidopsis metabolite and specific signaling molecule that promotes anchor root formation. In this work, we investigated the biological activity of a synthetic isomer of anchorene, iso-anchorene, which can be derived from repeated carotenoid cleavage. We show that iso-anchorene is a growth inhibitor that specifically inhibits primary root growth by reducing cell division rates in the root apical meristem. Using auxin efflux transporter marker lines, we also show that the effect of iso-anchorene on primary root growth involves the modulation of auxin homeostasis. Moreover, by using liquid chromatography–mass spectrometry analysis, we demonstrate that iso-anchorene is a natural Arabidopsis metabolite. Chemical inhibition of carotenoid biosynthesis led to a significant decrease in the iso-anchorene level, indicating that it originates from this metabolic pathway. Taken together, our results reveal a novel carotenoid-derived regulatory metabolite with a specific biological function that affects root growth, manifesting the biological importance of diapocarotenoids.     

The Rop GTPase switch controls multiple developmental processes in Arabidopsis

G proteins are universal molecular switches in eukaryotic signal transduction. The Arabidopsis genome sequence reveals no RAS small GTPase and only one or a few heterotrimeric G proteins, two predominant classes of signaling G proteins found in animals. In contrast, Arabidopsis possesses a unique family of 11 Rop GTPases that belong to the Rho family of small GTPases. Previous studies indicate that Rop controls actin-dependent pollen tube growth and H(2)O(2)-dependent defense responses. In this study, we tested the hypothesis that the Rop GTPase acts as a versatile molecular switch in signaling to multiple developmental processes in Arabidopsis. Immunolocalization using a general antibody against the Rop family proteins revealed a ubiquitous distribution of Rop proteins in all vegetative and reproductive tissues and cells in Arabidopsis. The cauliflower mosaic virus 35S promoter-directed expression of constitutively active GTP-bound rop2 (CA-rop2) and dominant negative GDP-bound rop2 (DN-rop2) mutant genes impacted many aspects of plant growth and development, including embryo development, seed dormancy, seedling development, lateral root initiation, morphogenesis of lateral organs in the shoot, shoot apical dominance and growth, phyllotaxis, and lateral organ orientation. The rop2 transgenic plants also displayed altered responses to the exogenous application of several hormones, such as abscisic acid-mediated seed dormancy, auxin-dependent lateral shoot initiation, and brassinolide-mediated hypocotyl elongation. CA-rop2 and DN-rop2 expression had opposite effects on most of the affected processes, supporting a direct signaling role for Rop in regulating these processes. Based on these observations and previous results, we propose that Rop2 and other members of the Rop family participate in multiple distinct signaling pathways that control plant growth, development, and responses to the environment.     

Environmental Regulation of Lateral Root Initiation in Arabidopsis

Plant morphology is dramatically influenced by environmental signals. The growth and development of the root system is an excellent example of this developmental plasticity. Both the number and placement of lateral roots are highly responsive to nutritional cues. This indicates that there must be a signal transduction pathway that interprets complex environmental conditions and makes the "decision" to form a lateral root at a particular time and place. Lateral roots originate from differentiated cells in adult tissues. These cells must reenter the cell cycle, proliferate, and redifferentiate to produce all of the cell types that make up a new organ. Almost nothing is known about how lateral root initiation is regulated or coordinated with growth conditions. Here, we report a novel growth assay that allows this regulatory mechanism to be dissected in Arabidopsis. When Arabidopsis seedlings are grown on nutrient media with a high sucrose to nitrogen ratio, lateral root initiation is dramatically repressed. Auxin localization appears to be a key factor in this nutrient-mediated repression of lateral root initiation. We have isolated a mutant, lateral root initiation 1 (lin1), that overcomes the repressive conditions. This mutant produces a highly branched root system on media with high sucrose to nitrogen ratios. The lin1 phenotype is specific to these growth conditions, suggesting that the lin1 gene is involved in coordinating lateral root initiation with nutritional cues. Therefore, these studies provide novel insights into the mechanisms that regulate the earliest steps in lateral root initiation and that coordinate plant development with the environment.     

Family-wide evaluation of RAPID ALKALINIZATION FACTOR peptides

Plant peptide hormones are important players that control various aspects of the lives of plants. RAPID ALKALINIZATION FACTOR (RALF) peptides have recently emerged as important players in multiple physiological processes. Numerous studies have increased our understanding of the evolutionary processes that shaped the RALF family of peptides. Nevertheless, to date, there is no comprehensive, family-wide functional study on RALF peptides. Here, we analyzed the phylogeny of the proposed multigenic RALF peptide family in the model plant Arabidopsis (Arabidopsis thaliana), ecotype Col-0, and tested a variety of physiological responses triggered by RALFs. Our phylogenetic analysis reveals that two of the previously proposed RALF peptides are not genuine RALF peptides, which leads us to propose a revision to the consensus AtRALF peptide family annotation. We show that the majority of AtRALF peptides, when applied exogenously as synthetic peptides, induce seedling or root growth inhibition and modulate reactive oxygen species (ROS) production in Arabidopsis. Moreover, our findings suggest that alkalinization and growth inhibition are, generally, coupled characteristics of RALF peptides. Additionally, we show that for the majority of the peptides, these responses are genetically dependent on FERONIA, suggesting a pivotal role for this receptor kinase in the perception of multiple RALF peptides.

Synthetic RAPID ALKALINIZATION FACTOR peptides induced a variety of physiological responses, many of which depend on FERONIA, a receptor kinase with functions in development and defense