Expression Profiling of Cytokinin Action in Arabidopsis

The phytohormone cytokinin is an important regulator of plant growth and development; however, relatively few genes that mediate cytokinin responses have been identified. Genome-wide analyses of Arabidopsis seedlings using the approximately 8,300-element Affymetrix Arabidopsis GeneChips (Affymetrix, Santa Clara, CA) to examine cytokinin-responsive genes were conducted, revealing at least 30 genes whose steady-state level of mRNA was elevated and at least 40 that were down-regulated at multiple time points after application of cytokinin. The cytokinin up-regulated genes include the type-A Arabidopsis response regulators (ARRs), which had been shown previously to be cytokinin primary response genes, cytokinin oxidase, which encodes an enzyme that degrades cytokinins, and several transcription factors. Cytokinin down-regulated genes include several peroxidases and kinases and an E3 ubiquitin ligase. We identified a common sequence motif enriched in the upstream regions of the most consistently cytokinin up-regulated genes. This motif is highly similar to the optimal DNA-binding sites for ARR1/ARR2, type-B ARRs that have been implicated in the transcriptional elevation of the type-A ARRs. Additionally, genome-wide analyses of cytokinin receptor mutants (wol/cre1) revealed large-scale changes in gene expression, including down-regulation of the type-A ARRs and several meristem and cell cycle genes, such as CycD3. Mutations in CRE1 reduced but did not eliminate the effect of cytokinin on gene expression for a subset of cytokinin-responsive genes and had little or no effect on others, suggesting functional redundancy among the cytokinin receptors

Cytokinin response factor 6 represses cytokinin-associated genes during oxidative stress

Cytokinin is a phytohormone that is well known for its roles in numerous plant growth and developmental processes, yet it has also been linked to abiotic stress response in a less defined manner. Arabidopsis (Arabidopsis thaliana) Cytokinin Response Factor 6 (CRF6) is a cytokinin-responsive AP2/ERF-family transcription factor that, through the cytokinin signaling pathway, plays a key role in the inhibition of dark-induced senescence. CRF6 expression is also induced by oxidative stress, and here we show a novel function for CRF6 in relation to oxidative stress and identify downstream transcriptional targets of CRF6 that are repressed in response to oxidative stress. Analysis of transcriptomic changes in wild-type and crf6 mutant plants treated with H2O2 identified CRF6-dependent differentially expressed transcripts, many of which were repressed rather than induced. Moreover, many repressed genes also show decreased expression in 35S:CRF6 overexpressing plants. Together, these findings suggest that CRF6 functions largely as a transcriptional repressor. Interestingly, among the H2O2 repressed CRF6-dependent transcripts was a set of five genes associated with cytokinin processes: (signaling) ARR6, ARR9, ARR11, (biosynthesis) LOG7, and (transport) ABCG14. We have examined mutants of these cytokinin-associated target genes to reveal novel connections to oxidative stress. Further examination of CRF6-DNA interactions indicated that CRF6 may regulate its targets both directly and indirectly. Together, this shows that CRF6 functions during oxidative stress as a negative regulator to control this cytokinin-associated module of CRF6-dependent genes and establishes a novel connection between cytokinin and oxidative stress response

Two-Component Elements Mediate Interactions between Cytokinin and Salicylic Acid in Plant Immunity

Recent studies have revealed an important role for hormones in plant immunity. We are now beginning to understand the contribution of crosstalk among different hormone signaling networks to the outcome of plant–pathogen interactions. Cytokinins are plant hormones that regulate development and responses to the environment. Cytokinin signaling involves a phosphorelay circuitry similar to two-component systems used by bacteria and fungi to perceive and react to various environmental stimuli. In this study, we asked whether cytokinin and components of cytokinin signaling contribute to plant immunity. We demonstrate that cytokinin levels in Arabidopsis are important in determining the amplitude of immune responses, ultimately influencing the outcome of plant–pathogen interactions. We show that high concentrations of cytokinin lead to increased defense responses to a virulent oomycete pathogen, through a process that is dependent on salicylic acid (SA) accumulation and activation of defense gene expression. Surprisingly, treatment with lower concentrations of cytokinin results in increased susceptibility. These functions for cytokinin in plant immunity require a host phosphorelay system and are mediated in part by type-A response regulators, which act as negative regulators of basal and pathogen-induced SA–dependent gene expression. Our results support a model in which cytokinin up-regulates plant immunity via an elevation of SA–dependent defense responses and in which SA in turn feedback-inhibits cytokinin signaling. The crosstalk between cytokinin and SA signaling networks may help plants fine-tune defense responses against pathogens

Multiscale modelling of auxin transport in the plant-root elongation zone

In the root elongation zone of a plant, the hormone auxin moves in a polar manner due to active transport facilitated by spatially distributed influx and efflux carriers present on the cell membranes. To understand how the cell-scale active transport and passive diffusion combine to produce the effective tissue-scale flux, we apply asymptotic methods to a cell-based model of auxin transport to derive systematically a continuum description from the spatially discrete one. Using biologically relevant parameter values, we show how the carriers drive the dominant tissue-scale auxin flux and we predict how the overall auxin dynamics are affected by perturbations to these carriers, for example, in knockout mutants. The analysis shows how the dominant behaviour depends on the cells' lengths, and enables us to assess the relative importance of the diffusive auxin flux through the cell wall. Other distinguished limits are also identified and their potential roles discussed. As well as providing insight into auxin transport, the study illustrates the use of multiscale (cell to tissue) methods in deriving simplified models that retain the essential biology and provide understanding of the underlying dynamics

Genome-wide identification and phylogenetic analysis of the ERF gene family in cucumbers

Members of the ERF transcription-factor family participate in a number of biological processes, viz., responses to hormones, adaptation to biotic and abiotic stress, metabolism regulation, beneficial symbiotic interactions, cell differentiation and developmental processes. So far, no tissue-expression profile of any cucumber ERF protein has been reported in detail. Recent completion of the cucumber full-genome sequence has come to facilitate, not only genome-wide analysis of ERF family members in cucumbers themselves, but also a comparative analysis with those in Arabidopsis and rice. In this study, 103 hypothetical ERF family genes in the cucumber genome were identified, phylogenetic analysis indicating their classification into 10 groups, designated I to X. Motif analysis further indicated that most of the conserved motifs outside the AP2/ERF domain, are selectively distributed among the specific clades in the phylogenetic tree. From chromosomal localization and genome distribution analysis, it appears that tandem-duplication may have contributed to CsERF gene expansion. Intron/exon structure analysis indicated that a few CsERFs still conserved the former intron-position patterns existent in the common ancestor of monocots and eudicots. Expression analysis revealed the widespread distribution of the cucumber ERF gene family within plant tissues, thereby implying the probability of their performing various roles therein. Furthermore, members of some groups presented mutually similar expression patterns that might be related to their phylogenetic groups

Auxin Response Factor2 (ARF2) and Its Regulated Homeodomain Gene HB33 Mediate Abscisic Acid Response in Arabidopsis

The phytohormone abscisic acid (ABA) is an important regulator of plant development and response to environmental stresses. In this study, we identified two ABA overly sensitive mutant alleles in a gene encoding Auxin Response Factor2 (ARF2). The expression of ARF2 was induced by ABA treatment. The arf2 mutants showed enhanced ABA sensitivity in seed germination and primary root growth. In contrast, the primary root growth and seed germination of transgenic plants over-expressing ARF2 are less inhibited by ABA than that of the wild type. ARF2 negatively regulates the expression of a homeodomain gene HB33, the expression of which is reduced by ABA. Transgenic plants over-expressing HB33 are more sensitive, while transgenic plants reducing HB33 by RNAi are more resistant to ABA in the seed germination and primary root growth than the wild type. ABA treatment altered auxin distribution in the primary root tips and made the relative, but not absolute, auxin accumulation or auxin signal around quiescent centre cells and their surrounding columella stem cells to other cells stronger in arf2-101 than in the wild type. These results indicate that ARF2 and HB33 are novel regulators in the ABA signal pathway, which has crosstalk with auxin signal pathway in regulating plant growth

Role of the Arabidopsis PIN6 auxin transporter in auxin homeostasis and auxin-mediated development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.Christopher I. Cazzonelli, Marleen Vanstraelen, Sibu Simon, Kuide Yin, Ashley Carron-Arthur, Nazia Nisar, Gauri Tarle, Abby J. Cuttriss¤, Iain R. Searle, Eva Benkova, Ulrike Mathesius, Josette Masle, Jiří Friml, Barry J. Pogso

Attenuated Food Anticipatory Activity and Abnormal Circadian Locomotor Rhythms in Rgs16 Knockdown Mice

Regulators of G protein signaling (RGS) are a multi-functional protein family, which functions in part as GTPase-activating proteins (GAPs) of G protein α-subunits to terminate G protein signaling. Previous studies have demonstrated that the Rgs16 transcripts exhibit robust circadian rhythms both in the suprachiasmatic nucleus (SCN), the master circadian light-entrainable oscillator (LEO) of the hypothalamus, and in the liver. To investigate the role of RGS16 in the circadian clock in vivo, we generated two independent transgenic mouse lines using lentiviral vectors expressing short hairpin RNA (shRNA) targeting the Rgs16 mRNA. The knockdown mice demonstrated significantly shorter free-running period of locomotor activity rhythms and reduced total activity as compared to the wild-type siblings. In addition, when feeding was restricted during the daytime, food-entrainable oscillator (FEO)-driven elevated food-anticipatory activity (FAA) observed prior to the scheduled feeding time was significantly attenuated in the knockdown mice. Whereas the restricted feeding phase-advanced the rhythmic expression of the Per2 clock gene in liver and thalamus in the wild-type animals, the above phase shift was not observed in the knockdown mice. This is the first in vivo demonstration that a common regulator of G protein signaling is involved in the two separate, but interactive circadian timing systems, LEO and FEO. The present study also suggests that liver and/or thalamus regulate the food-entrained circadian behavior through G protein-mediated signal transduction pathway(s)