Dennis E. Mathews

Multiple Type-B Response Regulators Mediate Cytokinin Signal Transduction in Arabidopsis

Type-B Arabidopsis thaliana response regulators (ARRs) are transcription factors that function in the final step of two-component signaling systems. To characterize their role in plant growth and development, we isolated T-DNA insertions within six of the genes (ARR1, ARR2, ARR10, ARR11, ARR12, and ARR18) from the largest subfamily of type-B ARRs and also constructed various double and triple combinations of these mutations. Higher order mutants revealed progressively decreased sensitivity to cytokinin, including effects on root elongation, lateral root formation, callus induction and greening, and induction of cytokinin primary response genes. The triple mutant arr1,10,12 showed almost complete insensitivity to cytokinin under many of the assay conditions used. By contrast, no significant change in the sensitivity to ethylene was found among the mutants examined. These results indicate that there is functional overlap among the type-B ARRs and that they act as positive regulators of cytokinin signal transduction.

Type B Response Regulators of Arabidopsis Play Key Roles in Cytokinin Signaling and Plant Development

The type B Arabidopsis Response Regulators (ARRs) of Arabidopsis thaliana are transcription factors that act as positive regulators in the two-component cytokinin signaling pathway. We employed a mutant-based approach to perform a detailed characterization of the roles of ARR1, ARR10, and ARR12 in plant growth and development. The most pronounced phenotype was found in the arr1-3 arr10-5 arr12-1 triple loss-of-function mutant, which showed almost complete insensitivity to high levels of exogenously applied cytokinins. The triple mutant exhibited reduced stature due to decreased cell division in the shoot, enhanced seed size, increased sensitivity to light, altered chlorophyll and anthocyanin concentrations, and an aborted primary root with protoxylem but no metaxylem. Microarray analysis revealed that expression of the majority of cytokinin-regulated genes requires the function of ARR1, ARR10, and ARR12. Characterization of double mutants revealed differing contributions of the type B ARRs to mutant phenotypes. Our results support a model in which cytokinin regulates a wide array of downstream responses through the action of a multistep phosphorelay that culminates in transcriptional regulation by ARR1, ARR10, and ARR12.

Type-B response regulators display overlapping expression patterns in Arabidopsis

Two-component signaling systems, involving His kinases, His-containing phosphotransfer proteins, and response regulators, have been implicated in plant responses to hormones and environmental factors. Genomic analysis of Arabidopsis supports the existence of 22 response regulators (ARRs) that can be divided into at least two distinct groups designated type-A and type-B. Phylogenetic analysis indicates that the type-B family is composed of one major and two minor subfamilies. The expression of the type-B ARRs was examined by using both reverse transcription-PCR and β-glucuronidase fusion constructs. The major subfamily of type-B ARRs showed particularly high expression in regions where cytokinins play a significant role, including cells in the apical meristem region and in young leaves that would be undergoing cell division. Multiple members within this same subfamily of type-B ARRs were expressed near the root tip with highest expression in the root elongation zone. β-Glucuronidase-fusions to full-length ARR2, ARR12, and ARR19 were nuclear localized, consistent with a role in transcriptional regulation. These data suggest that differing expression levels of the type-B ARRs may play a role in modulating the cellular responses to cytokinin.

Effect of Ethylene Pathway Mutations upon Expression of the Ethylene Receptor ETR1 from Arabidopsis

The ethylene receptor family of Arabidopsis consists of five members, one of these being ETR1. The effect of ethylene pathway mutations upon expression of ETR1 was examined. For this purpose, ETR1 levels were quantified in mutant backgrounds containing receptor loss-of-function mutations, ethylene-insensitive mutations, and constitutive ethylene response mutations. Ethylene-insensitive mutations of ETR1 resulted in a posttranscriptional increase in levels of the mutant receptor. Treatment of seedlings with silver, which leads to ethylene insensitivity, also resulted in an increase in levels of ETR1. Loss-of-function mutations of ETR1 resulted in both transcriptional and posttranscriptional changes in levels of the receptor. Most other ethylene pathway mutations, including a newly isolated T-DNA insertion mutation in the gene encoding the ethylene receptor ERS1, had relatively minor effects upon the expression of ETR1. Our results indicate that mutations in ETR1 can affect expression at the posttranscriptional level, and suggest that these posttranscriptional changes may contribute to the phenotypes observed in the mutants. Our results also refine the model on how mutations in ethylene receptors are able to confer dominant ethylene insensitivity upon plants.

Functional Characterization of Type-B Response Regulators in the Arabidopsis Cytokinin Response

Five out of 11 related transcription factors were found to mediate the cytokinin response based on complementation analysis of a cytokinin-signaling mutant. Cytokinins play critical roles in plant growth and development, with the transcriptional response to cytokinin being mediated by the type-B response regulators. In Arabidopsis (Arabidopsis thaliana), type-B response regulators (ARABIDOPSIS RESPONSE REGULATORS [ARRs]) form three subfamilies based on phylogenic analysis, with subfamily 1 having seven members and subfamilies 2 and 3 each having two members. Cytokinin responses are predominantly mediated by subfamily 1 members, with cytokinin-mediated effects on root growth and root meristem size correlating with type-B ARR expression levels. To determine which type-B ARRs can functionally substitute for the subfamily 1 members ARR1 or ARR12, we expressed different type-B ARRs from the ARR1 promoter and assayed their ability to rescue arr1 arr12 double mutant phenotypes. ARR1, as well as a subset of other subfamily 1 type-B ARRs, restore the cytokinin sensitivity to arr1 arr12. Expression of ARR10 from the ARR1 promoter results in cytokinin hypersensitivity and enhances shoot regeneration from callus tissue, correlating with enhanced stability of the ARR10 protein compared with the ARR1 protein. Examination of transfer DNA insertion mutants in subfamilies 2 and 3 revealed little effect on several well-characterized cytokinin responses. However, a member of subfamily 2, ARR21, restores cytokinin sensitivity to arr1 arr12 roots when expressed from the ARR1 promoter, indicating functional conservation of this divergent family member. Our results indicate that the type-B ARRs have diverged in function, such that some, but not all, can complement the arr1 arr12 mutant. In addition, our results indicate that type-B ARR expression profiles in the plant, along with posttranscriptional regulation, play significant roles in modulating their contribution to cytokinin signaling.

Cytokinin-dependent specification of the functional megaspore in the Arabidopsis female gametophyte

The life cycle of higher plants alternates between the diploid sporophytic and the haploid gametophytic phases. In angiosperms, male and female gametophytes develop within the sporophyte. During female gametophyte (FG) development, a single archesporial cell enlarges and differentiates into a megaspore mother cell, which then undergoes meiosis to give rise to four megaspores. In most species of higher plants, including Arabidopsis thaliana, the megaspore closest to the chalaza develops into the functional megaspore (FM), and the remaining three megaspores degenerate. Here, we examined the role of cytokinin signaling in FG development. We characterized the FG phenotype in three triple mutants harboring non-overlapping T-DNA insertions in cytokinin AHK receptors. We demonstrate that even the strongest mutant is not a complete null for the cytokinin receptors. Only the strongest mutant displayed a near fully penetrant disruption of FG development, and the weakest triple ahk mutant had only a modest FG phenotype. This suggests that cytokinin signaling is essential for FG development, but that only a low threshold of signaling activity is required for this function. Furthermore, we demonstrate that there is elevated cytokinin signaling localized in the chalaza of the ovule, which is enhanced by the asymmetric localization of cytokinin biosynthetic machinery and receptors. We show that an FM-specific marker is absent in the multiple ahk ovules, suggesting that disruption of cytokinin signaling elements in Arabidopsis blocks the FM specification. Together, this study reveals a chalazal-localized sporophytic cytokinin signal that plays an important role in FM specification in FG development.

Cytokinin acts through the auxin influx carrier AUX1 to regulate cell elongation in the root

Hormonal interactions are crucial for plant development. In Arabidopsis, cytokinins inhibit root growth through effects on cell proliferation and cell elongation. Here, we define key mechanistic elements in a regulatory network by which cytokinin inhibits root cell elongation in concert with the hormones auxin and ethylene. The auxin importer AUX1 functions as a positive regulator of cytokinin responses in the root; mutation of AUX1 specifically affects the ability of cytokinin to inhibit cell elongation but not cell proliferation. AUX1 is required for cytokinin-dependent changes of auxin activity in the lateral root cap associated with the control of cell elongation. Cytokinin regulates root cell elongation through ethylene-dependent and -independent mechanisms, both hormonal signals converging on AUX1 as a regulatory hub. An autoregulatory circuit is identified involving the control of ARR10 and AUX1 expression by cytokinin and auxin, this circuit potentially functioning as an oscillator to integrate the effects of these two hormones. Taken together, our results uncover several regulatory circuits controlling interactions of cytokinin with auxin and ethylene, and support a model in which cytokinin regulates shootward auxin transport to control cell elongation and root growth. Highlighted article: A model for how cytokinin inhibits Arabidopsis root cell elongation, working in concert with the hormones auxin and ethylene, is put forward.

Molecular analysis of the hull-less seed trait in pumpkin: expression profiles of genes related to seed coat development11

We undertook a comparative study of molecular changes during development of seed coats in the wild-type and a recessive hull-less mutant of pumpkin ( Cucurbita pepo L.), with the goal of identifying key genes involved in secondary cell wall development in the testa. The mature mutant testa has reduced amounts of cellulose and lignin as compared to the wild type. The expression patterns of several genes involved in secondary cell wall biosynthesis during the development of the testa are described. These genes are: CELLULOSE SYNTHASE , PHENYLALANINE AMMONIA-LYASE , 4-COUMARATE-CoA LIGASE , and CINNAMOYL-CoA REDUCTASE . Additionally, the expression patterns of a few genes that were differentially expressed in the two genotypes during testa development ( GLUTATHIONE REDUCTASE , ABSCISIC ACID RESPONSE PROTEIN E , a SERINE-THREONINE KINASE , and a β - UREIDOPROPIONASE ) are presented. The results show a coordinated expression of several genes involved in cellulose and lignin biosynthesis, as well as marked differences in the level of their expression between the two genotypes during testa development. There is generally a higher expression of genes involved in cellulose and lignin biosynthesis in the wild-type testa as compared to the mutant. The molecular data presented here are consistent with anatomical and biochemical differences between the wild-type and the mutant testae. An understanding of the genes involved in cell wall development in the testa will facilitate the manipulation of seed coat development in Cucurbita and other species for diverse commercial applications.

A role for two-component signaling elements in the Arabidopsis growth recovery response to ethylene

Abstract Previous studies indicate that the ability of Arabidopsis seedlings to recover normal growth following an ethylene treatment involves histidine kinase activity of the ethylene receptors. As histidine kinases can function as inputs for a two‐component signaling system, we examined loss‐of‐function mutants involving two‐component signaling elements. We find that mutants of phosphotransfer proteins and type‐B response regulators exhibit a defect in their ethylene growth recovery response similar to that found with the loss‐of‐function ethylene receptor mutant etr1‐7. The ability of two‐component signaling elements to regulate the growth recovery response to ethylene functions independently from their well‐characterized role in cytokinin signaling, based on the analysis of cytokinin receptor mutants as well as following chemical inhibition of cytokinin biosynthesis. Histidine kinase activity of the receptor ETR1 also facilitates growth recovery in the ethylene hypersensitive response, which is characterized by a transient decrease in growth rate when seedlings are treated continuously with a low dose of ethylene; however, this response was found to operate independently of the type‐B response regulators. These results indicate that histidine kinase activity of the ethylene receptor ETR1 performs two independent functions: (a) regulating the growth recovery to ethylene through a two‐component signaling system involving phosphotransfer proteins and type‐B response regulators and (b) regulating the hypersensitive response to ethylene in a type‐B response regulator independent manner.

Molecular characterization of four genes involved in sulfur metabolism in Porphyra purpurea (Roth) C. Agardh

Sulfated polysaccharides (carrageenans and agars) are among the most important products of red algae that are used as food additives as well as in molecular biology research. The quality and value of the product is greatly dependent on the levels and sites of sulfation of the polysaccharides. Little information is currently available on the molecular details of sulfur metabolism in red algae. Considering the economic importance of sulfated polysaccharide, elucidating the molecular details of sulfur metabolism in these organisms could help in future endeavors to improve algal commercial value, e.g., through genetic engineering. A cDNA library from the red alga Porphyra purpurea (Roth) C. Agardh was used to isolate four cDNAs with homology to genes encoding known sulfur assimilation enzymes: sulfate adenyltransferase (ATP sulfurylase), adenosine 5′-phosphosulfate kinase (APSK), sulfite reductase, and cysteine synthase. These cDNAs were characterized with respect to their molecular properties and a cDNA with homology to APSK was used to functionally complement an Escherichia coli auxotroph APSK− mutant. The other cDNAs are being similarly characterized with respect to their ability to produce functional enzymes. Elucidation of the regulation of expression of these genes will aid in future research to determine the biochemical and genetic details of the sulfate assimilation pathway as well as its genetic manipulation in red algae.