Michael G. Mason

Type-A Arabidopsis Response Regulators Are Partially Redundant Negative Regulators of Cytokinin Signaling

Type-A Arabidopsis (Arabidopsis thaliana) response regulators (ARRs) are a family of 10 genes that are rapidly induced by cytokinin and are highly similar to bacterial two-component response regulators. We have isolated T-DNA insertions in six of the type-A ARRs and constructed multiple insertional mutants, including the arr3,4,5,6,8,9 hextuple mutant. Single arr mutants were indistinguishable from the wild type in various cytokinin assays; double and higher order arr mutants showed progressively increasing sensitivity to cytokinin, indicating functional overlap among type-A ARRs and that these genes act as negative regulators of cytokinin responses. The induction of cytokinin primary response genes was amplified in arr mutants, indicating that the primary response to cytokinin is affected. Spatial patterns of ARR gene expression were consistent with partially redundant function of these genes in cytokinin signaling. The arr mutants show altered red light sensitivity, suggesting a general involvement of type-A ARRs in light signal transduction. Further, morphological phenotypes of some arr mutants suggest complex regulatory interactions and gene-specific functions among family members.

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.

Strigolactones Suppress Adventitious Rooting in Arabidopsis and Pea

Adventitious root formation is essential for the propagation of many commercially important plant species and involves the formation of roots from nonroot tissues such as stems or leaves. Here, we demonstrate that the plant hormone strigolactone suppresses adventitious root formation in Arabidopsis (Arabidopsis thaliana) and pea (Pisum sativum). Strigolactone-deficient and response mutants of both species have enhanced adventitious rooting. CYCLIN B1 expression, an early marker for the initiation of adventitious root primordia in Arabidopsis, is enhanced in more axillary growth2 (max2), a strigolactone response mutant, suggesting that strigolactones restrain the number of adventitious roots by inhibiting the very first formative divisions of the founder cells. Strigolactones and cytokinins appear to act independently to suppress adventitious rooting, as cytokinin mutants are strigolactone responsive and strigolactone mutants are cytokinin responsive. In contrast, the interaction between the strigolactone and auxin signaling pathways in regulating adventitious rooting appears to be more complex. Strigolactone can at least partially revert the stimulatory effect of auxin on adventitious rooting, and auxin can further increase the number of adventitious roots in max mutants. We present a model depicting the interaction of strigolactones, cytokinins, and auxin in regulating adventitious root formation.

Sugar demand, not auxin, is the initial regulator of apical dominance

Significance It is commonly accepted that the plant hormone auxin mediates apical dominance. However, we have discovered that apical dominance strongly correlates with sugar availability and not apically supplied auxin. We have revealed that apical dominance is predominantly controlled by the shoot tip’s intense demand for sugars, which limits sugar availability to the axillary buds. These findings overturn a long-standing hypothesis on apical dominance and encourage us to reevaluate the relationship between hormones and sugars in this and other aspects of plant development. For almost a century the plant hormone auxin has been central to theories on apical dominance, whereby the growing shoot tip suppresses the growth of the axillary buds below. According to the classic model, the auxin indole-3-acetic acid is produced in the shoot tip and transported down the stem, where it inhibits bud growth. We report here that the initiation of bud growth after shoot tip loss cannot be dependent on apical auxin supply because we observe bud release up to 24 h before changes in auxin content in the adjacent stem. After the loss of the shoot tip, sugars are rapidly redistributed over large distances and accumulate in axillary buds within a timeframe that correlates with bud release. Moreover, artificially increasing sucrose levels in plants represses the expression of BRANCHED1 (BRC1), the key transcriptional regulator responsible for maintaining bud dormancy, and results in rapid bud release. An enhancement in sugar supply is both necessary and sufficient for suppressed buds to be released from apical dominance. Our data support a theory of apical dominance whereby the shoot tip’s strong demand for sugars inhibits axillary bud outgrowth by limiting the amount of sugar translocated to those buds.

Heterotrimeric G Protein γ Subunits Provide Functional Selectivity in Gβγ Dimer Signaling in Arabidopsis

The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Gα and Gβ subunit genes and two Gγ subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Gγ genes revealed specialized roles for each Gγ subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1- and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that Gβγ1 and Gβγ2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Gγ subunit in this root response differs, with Gβγ1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gβγ2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in Gβγ signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both Gγ subunits form functional Gβγ dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein–mediated signaling in plants.

Isolation of a novel G-protein γ-subunit from Arabidopsis thaliana and its interaction with Gβ

There is increasing evidence that heterotrimeric G-proteins (G-proteins) are involved in many plant processes including phytohormone response, pathogen defence and stomatal control. In animal systems, each of the three G-protein subunits belong to large multigene families; however, few subunits have been isolated from plants. Here we report the cloning of a second plant G-protein γ-subunit (AGG2) from Arabidopsis thaliana. The predicted AGG2 protein sequence shows 48% identity to the first identified Arabidopsis Gγ-subunit, AGG1. Furthermore, AGG2 contains all of the conserved characteristics of γ-subunits including a small size (100 amino acids, 11.1 kDa), C-terminal CAAX box and a N-terminal α-helix region capable of forming a coiled-coil interaction with the β-subunit. A strong interaction between AGG2 and both the tobacco (TGB1) and Arabidopsis (AGB1) β-subunits was observed in vivo using the yeast two-hybrid system. The strong association between AGG2 and AGB1 was confirmed in vitro. Southern and Northern analyses showed that AGG2 is a single copy gene in Arabidopsis producing two transcripts that are present in all tissues tested. The isolation of a second γ-subunit from A. thaliana indicates that plant G-proteins, like their mammalian counterparts, may form different heterotrimer combinations that presumably regulate multiple signal transduction pathways.

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.

Type-B response regulators ARR1 and ARR12 regulate expression of AtHKT1;1 and accumulation of sodium in Arabidopsis shoots

Soil salinity affects a large proportion of the land worldwide, forcing plants to evolve a number of mechanisms to cope with salt stress. Cytokinin plays a role in the plant response to salt stress, but little is known about the mechanism by which cytokinin controls this process. We used a molecular genetics approach to examine the influence of cytokinin on sodium accumulation and salt sensitivity in Arabidopsis thaliana. Cytokinin application was found to increase sodium accumulation in the shoots of Arabidopsis, but had no significant affect on the sodium content in the roots. Consistent with this, altered sodium accumulation phenotypes were observed in mutants of each gene class of the cytokinin signal transduction pathway, including receptors, phospho-transfer proteins, and type-A and type-B response regulators. Expression of the gene encoding Arabidopsis high-affinity K(+) transporter 1;1 (AtHKT1;1), a gene responsible for removing sodium ions from the root xylem, was repressed by cytokinin treatment, but showed significantly elevated expression in the cytokinin response double mutant arr1-3 arr12-1. Our data suggest that cytokinin, acting through the transcription factors ARR1 and ARR12, regulates sodium accumulation in the shoots by controlling the expression of AtHKT1;1 in the roots.

Lateral branching oxidoreductase acts in the final stages of strigolactone biosynthesis in Arabidopsis

Significance Strigolactone hormones regulate many plant growth and developmental processes and are particularly important in regulating growth in response to nonoptimal conditions. Plants produce a range of bioactive strigolactone-like compounds, suggesting that the biosynthesis pathway is complex. Despite this complexity, only one type of enzyme, the MORE AXILLARY GROWTH1 (MAX1) cytochrome P450, has been attributed to the diversity of strigolactones. Using transcriptomics and reverse genetics, we discovered a previously uncharacterized gene that encodes a 2-oxoglutarate and Fe(II)-dependent dioxygenase involved in strigolactone production downstream of MAX1. Studies with the corresponding mutant have shown that previously identified strigolactone-type compounds in Arabidopsis are not the major strigolactone-type shoot branching hormone in this model species. Strigolactones are a group of plant compounds of diverse but related chemical structures. They have similar bioactivity across a broad range of plant species, act to optimize plant growth and development, and promote soil microbe interactions. Carlactone, a common precursor to strigolactones, is produced by conserved enzymes found in a number of diverse species. Versions of the MORE AXILLARY GROWTH1 (MAX1) cytochrome P450 from rice and Arabidopsis thaliana make specific subsets of strigolactones from carlactone. However, the diversity of natural strigolactones suggests that additional enzymes are involved and remain to be discovered. Here, we use an innovative method that has revealed a missing enzyme involved in strigolactone metabolism. By using a transcriptomics approach involving a range of treatments that modify strigolactone biosynthesis gene expression coupled with reverse genetics, we identified LATERAL BRANCHING OXIDOREDUCTASE (LBO), a gene encoding an oxidoreductase-like enzyme of the 2-oxoglutarate and Fe(II)-dependent dioxygenase superfamily. Arabidopsis lbo mutants exhibited increased shoot branching, but the lbo mutation did not enhance the max mutant phenotype. Grafting indicated that LBO is required for a graft-transmissible signal that, in turn, requires a product of MAX1. Mutant lbo backgrounds showed reduced responses to carlactone, the substrate of MAX1, and methyl carlactonoate (MeCLA), a product downstream of MAX1. Furthermore, lbo mutants contained increased amounts of these compounds, and the LBO protein specifically converts MeCLA to an unidentified strigolactone-like compound. Thus, LBO function may be important in the later steps of strigolactone biosynthesis to inhibit shoot branching in Arabidopsis and other seed plants.