Growth promotion of Chinese cabbage and Arabidopsis by Piriformospora indica is not stimulated by mycelium-synthesized auxin

Piriformospora indica, an endophytic fungus of the order Sebacinales, interacts with the roots of a large variety of plant species. We compared the interaction of this fungus with Chinese cabbage (Brassica campestris subsp. chinensis) and Arabidopsis seedlings. The development of shoots and roots of Chinese cabbage seedlings was strongly promoted by P. indica and the fresh weight of the seedlings increased approximately twofold. The strong stimulation of root hair development resulted in a bushy root phenotype. The auxin level in the infected Chinese cabbage roots was twofold higher compared with the uncolonized controls. Three classes of auxin-related genes, which were upregulated by P. indica in Chinese cabbage roots, were isolated from a double-subtractive expressed sequence tag library: genes for proteins related to cell wall acidification, intercellular auxin transport carrier proteins such as AUX1, and auxin signal proteins. Overexpression of B. campestris BcAUX1 in Arabidopsis strongly promoted growth and biomass production of Arabidopsis seedlings and plants; the roots were highly branched but not bushy when compared with colonized Chinese cabbage roots. This suggests that BcAUX1 is a target of P. indica in Chinese cabbage. P. indica also promoted growth of Arabidopsis seedlings but the auxin levels were not higher and auxin genes were not upregulated, implying that auxin signaling is a more important target of P. indica in Chinese cabbage than in Arabidopsis. The fungus also stimulated growth of Arabidopsis aux1 and aux1/axr4 and rhd6 seedlings. Furthermore, a component in an exudate fraction from P. indica but not auxin stimulated growth of Chinese cabbage and Arabidopsis seedlings. We propose that activation of auxin biosynthesis and signaling in the roots might be the cause for the P. indica-mediated growth phenotype in Chinese cabbage.     

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.     

A leucine-rich repeat protein is required for growth promotion and enhanced seed production mediated by the endophytic fungus Piriformospora indica in Arabidopsis thaliana

Piriformospora indica, a basidiomycete of the Sebacinaceae family, promotes the growth, development and seed production of a variety of plant species. Arabidopsis plants colonized with the fungus produce 22% more seeds than uncolonized plants. Deactivating the Arabidopsis single-copy gene DMI-1, which encodes an ion carrier required for mycorrihiza formation in legumes, does not affect the beneficial interaction between the two symbiotic partners. We used cellular and molecular responses initiated during the establishment of the interaction between P. indica and Arabidopsis roots to isolate mutants that fail to respond to the fungus. An ethylmethane sulfonate mutant (Piriformospora indica-insensitive-2; pii-2), and a corresponding insertion line, are impaired in a leucine-rich repeat protein (At1g13230). The protein pii-2, which contains a putative endoplasmic reticulum retention signal, is also found in Triton X-100-insoluble plasma membrane microdomains, suggesting that it is present in the endoplasmic reticulum/plasma membrane continuum in Arabidopsis roots. The microdomains also contain an atypical receptor protein (At5g16590) containing leucine-rich repeats, the message of which is transiently upregulated in Arabidopsis roots in response to P. indica. This response is not detectable in At1g13230 mutants, and the protein is not detectable in the At1g13230 mutant microdomains. Partial deactivation of a gene for a sphingosine kinase, which is required for the biosynthesis of sphingolipid found in plasma membrane microdomains, also affects the Arabidopsis/P. indica interaction. Thus, pii-2, and presumably also At5g16590, two proteins present in plasma membrane microdomains, appear to be involved in P. indica-induced growth promotion and enhanced seed production in Arabidopsis thaliana.     

Indole-3-acetaldoxime-derived compounds restrict root colonization in the beneficial interaction between Arabidopsis roots and the endophyte Piriformospora indica

The growth-promoting and root-colonizing endophyte Piriformospora indica induces camalexin and the expression of CYP79B2, CYP79B3, CYP71A13, PAD3, and WRKY33 required for the synthesis of indole-3-acetaldoxime (IAOx)-derived compounds in the roots of Arabidopsis seedlings. Upregulation of the mRNA levels by P. indica requires cytoplasmic calcium elevation and mitogen-activated protein kinase 3 but not root-hair-deficient 2, radical oxygen production, or the 3-phosphoinositide-dependent kinase 1/oxidative signal-inducible 1 pathway. Because P. indica-mediated growth promotion is impaired in cyp79B2 cyp79B3 seedlings, while pad3 seedlings-which do not accumulate camalexin-still respond to the fungus, IAOx-derived compounds other than camalexin (e.g., indole glucosinolates) are required during early phases of the beneficial interaction. The roots of cyp79B2 cyp79B3 seedlings are more colonized than wild-type roots, and upregulation of the defense genes pathogenesis-related (PR)-1, PR-3, PDF1.2, phenylalanine ammonia lyase, and germin indicates that the mutant responds to the lack of IAOx-derived compounds by activating other defense processes. After 6 weeks on soil, defense genes are no longer upregulated in wild-type, cyp79B2 cyp79B3, and pad3 roots. This results in uncontrolled fungal growth in the mutant roots and reduced performance of the mutants. We propose that a long-term harmony between the two symbionts requires restriction of root colonization by IAOx-derived compounds.     

The Cytokinin-hypersensitive genes of Arabidopsis negatively regulate the cytokinin-signaling pathway for cell division and chloroplast development

We isolated Arabidopsis thaliana mutants that respond more sensitively than the wild type to cytokinins. The calli produced from the mutants exhibit typical cytokinin responses, including rapid proliferation and chloroplast development in response to lower levels of cytokinins than in the wild type. The mutations are recessive and belong to two complementation groups designated ckh1 and ckh2 for cytokinin-hypersensitive. CKH1 and CKH2 were mapped to the top of chromosome I and the middle of chromosome II, respectively. The cytokinin levels in these mutants were not increased. We speculate that the CKH1 and CKH2 gene products negatively regulate the signaling pathway leading from cytokinin perception to cell proliferation and chloroplast development.     

Effect of Trichoderma Colonization on Auxin-Mediated Regulation of Root Elongation

The biocontrol fungus Trichoderma harzianum 1295-22 increases root growth in addition to roles in suppressing disease. Its agricultural use could be expanded if the mechanism of growth enhancement were known. Among the proposed mechanisms of growth enhancement is that the fungus counteracts auxin inhibition of root-cell elongation. We tested whether there was evidence for a secreted auxin inhibitor, for enhanced auxin degradation, or for altered auxin sensitivity. Our results provide no support for any of these mechanisms. Trichoderma secretions inhibited growth, whereas an auxin inhibitor would increase growth. Auxin inhibited growth to the same extent in colonized and uncolonized roots, indicating no change in auxin sensitivity. Endogenous auxin levels maintained growth closer to the maximum in uncolonized roots, indicating stronger auxin limitation of growth in colonized roots. These tests indicated that Trichoderma-colonized roots had a faster maximum growth rate, but an unchanged response to auxin.     

Analysis of gametophytic development in the moss,Physcomitrella patens,using auxin and cytokinin resistant mutants

Mutants altered in their response to auxins and cytokinins have been isolated in the moss Physcomitrella patens either by screening clones from mutagenized spores for growth on high concentrations of cytokinin or auxin, in which case mutants showing altered sensitivities can be recognized 3–4 weeks later, or by non-selective isolation of morphologically abnormal mutants, some of which are found to have altered sensitivities. Most of the mutants obtained selectively are also morphologically abnormal. The mutants are heterogeneous in their responses to auxin and cytokinin, and the behaviour of some is consistent with their being unable to make auxin, while that of others may be due to their being unable to synthesize cytokinin. Physiological analysis of the mutants has shown that both endogenous auxin and cytokinin are likely to play important and interdependent roles in several steps of gametophytic development. Although their morphological abnormalities lead to sterility, genetic analysis of some of the mutants has been possible by polyethyleneglycol induced protoplast fusion.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - NAA 1-naphthalene acetic acid - 2,4D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IAP 6-( ²isopentenyl) aminopurine - NAR NAA resistant mutants - BAR BAP resistant mutants     

AUXIN UP-REGULATED F-BOX PROTEIN1 regulates the cross talk between auxin transport and cytokinin signaling during plant root growth

Plant root development is mediated by the concerted action of the auxin and cytokinin phytohormones, with cytokinin serving as an antagonist of auxin transport. Here, we identify the AUXIN UP-REGULATED F-BOX PROTEIN1 (AUF1) and its potential paralog AUF2 as important positive modifiers of root elongation that tether auxin movements to cytokinin signaling in Arabidopsis (Arabidopsis thaliana). The AUF1 mRNA level in roots is strongly up-regulated by auxin but not by other phytohormones. Whereas the auf1 single and auf1 auf2 double mutant roots grow normally without exogenous auxin and respond similarly to the wild type upon auxin application, their growth is hypersensitive to auxin transport inhibitors, with the mutant roots also having reduced basipetal and acropetal auxin transport. The effects of auf1 on auxin movements may be mediated in part by the misexpression of several PIN-FORMED (PIN) auxin efflux proteins, which for PIN2 reduces its abundance on the plasma membrane of root cells. auf1 roots are also hypersensitive to cytokinin and have increased expression of several components of cytokinin signaling. Kinematic analyses of root growth and localization of the cyclin B mitotic marker showed that AUF1 does not affect root cell division but promotes cytokinin-mediated cell expansion in the elongation/differentiation zone. Epistasis analyses implicate the cytokinin regulator ARR1 or its effector(s) as the target of the SKP1-Cullin1-F Box (SCF) ubiquitin ligases assembled with AUF1/2. Given the wide distribution of AUF1/2-type proteins among land plants, we propose that SCF(AUF1/2) provides additional cross talk between auxin and cytokinin, which modifies auxin distribution and ultimately root elongation.     

Cytokinins act directly on lateral root founder cells to inhibit root initiation

In Arabidopsis thaliana, lateral roots are formed from root pericycle cells adjacent to the xylem poles. Lateral root development is regulated antagonistically by the plant hormones auxin and cytokinin. While a great deal is known about how auxin promotes lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to disrupt lateral root initiation and the regular pattern of divisions that characterizes lateral root development in Arabidopsis. To identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.     

Mutations at CRE1 impair cytokinin-induced repression of phosphate starvation responses in Arabidopsis

Plants display a number of responses to low phosphate availability, involving biochemical and developmental changes. Recently we have shown that many of these responses can be repressed in roots by exogenous addition of cytokinins. In order to understand the genetic basis to this effect of cytokinins, and its relation with the better known roles of cytokinins in the control of cell-cycle and differentiation, we have undertaken mutant screening and characterization using a transgenic line of Arabidopsis thaliana harbouring a reporter gene specifically responsive to Pi starvation (AtIPS1::GUS). One type of mutant identified displayed reduced sensitivity of AtIPS1::GUS to cytokinin repression. Several other Pi starvation response genes showed reduced cytokinin sensitivity in these lines. These mutants also showed reduced cytokinin repression of the anthocyanin accumulation induced by Pi starvation in the aerial part of the plants. Mapping and molecular characterization of these mutants showed that they were allelic of CRE1/WOL, a locus known to encode a cytokinin receptor. CRE1 is downregulated by Pi starvation and induced by cytokinins, both in the wild-type and in the cre1 mutants, in which cre1 mRNA levels are higher. These results reveal the existence of a positive feed-back loop, in addition to the already established negative feedback loop, in cytokinin signalling and indicate that the negative regulation of Pi starvation responses by cytokinins involves a two-component signalling circuitry, as it is the case of other types of cytokinin response.     

Mutations in the huge Arabidopsis gene BIG affect a range of hormone and light responses

In independent genetic screens, for shade-avoidance response and cytokinin sensitivity, we identified two Arabidopsis mutants, attenuated shade avoidance 1 (asa1) and umbrella1 (umb1), which have very similar pleiotropic phenotypes. asa1 and umb1 are allelic to tir3-1, and are caused by mutations in BIG, which is required for normal auxin efflux. They have a compact rosette, fewer lateral roots, delayed flowering, more secondary inflorescence, smaller seeds and, in the Laer-0 background, much shorter internodes between adjacent flowers, suggesting an interaction between BIG and ERECTA. These mutants have organ-specific defects in response to cytokinins, ethylene, N-1-naphthylphthalamic acid (NPA) and gibberellin (GA). The phenotype of the asa1 ga1-3 double mutant is consistent with defects in GA signalling. There are subtle effects in responses to auxins, abscisic acid and brassinolide. Elongation growth associated with shade avoidance in phyA phyB null mutants is suppressed by asa1 in all organs other than the hypocotyl. Therefore, we here provide evidence that BIG is a key player not just in auxin signalling, but in a multitude of light and hormone pathways.     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

Root development: cytokinin transport matters, too!

Unlike the plant hormone auxin, the mechanism and function of cytokinin transport is poorly characterised. Two new studies now demonstrate that cytokinins transported from shoot to roots via the phloem are critical for creating mutually exclusive auxin and cytokinin signalling domains that control root vascular patterning.     

Phloem-transported cytokinin regulates polar auxin transport and maintains vascular pattern in the root meristem

Cytokinin phytohormones regulate a variety of developmental processes in the root such as meristem size, vascular pattern, and root architecture [1-3]. Long-distance transport of cytokinin is supported by the discovery of cytokinins in xylem and phloem sap [4] and by grafting experiments between wild-type and cytokinin biosynthesis mutants [5]. Acropetal transport of cytokinin (toward the shoot apex) has also been implicated in the control of shoot branching [6]. However, neither the mode of transport nor a developmental role has been shown for basipetal transport of cytokinin (toward the root apex). In this paper, we combine the use of a new technology that blocks symplastic connections in the phloem with a novel approach to visualize radiolabeled hormones in planta to examine the basipetal transport of cytokinin. We show that this occurs through symplastic connections in the phloem. The reduction of cytokinin levels in the phloem leads to a destabilization of the root vascular pattern in a manner similar to mutants affected in auxin transport or cytokinin signaling [7]. Together, our results demonstrate a role for long-distance basipetal transport of cytokinin in controlling polar auxin transport and maintaining the vascular pattern in the root meristem.     

Cytokinin-induced hypocotyl elongation in light-grown<Emphasis Type="Italic"> Arabidopsis</Emphasis> plants with inhibited ethylene action or indole-3-acetic acid transport

Cytokinins inhibit hypocotyl elongation in darkness but have no obvious effect on hypocotyl length in the light. However, we found that cytokinins do promote hypocotyl elongation in the light when ethylene action is blocked. A 50% increase in Arabidopsis thaliana (L.) Heynh. hypocotyl length was observed in response to N⁶-benzyladenine (BA) treatment in the presence of Ag⁺. The level of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid was strongly increased, indicating that ethylene biosynthesis was up-regulated by treatment with cytokinin. Furthermore, the effects of cytokinins on hypocotyl elongation were also tested using a series of mutants in the cascade of the ethylene-signal pathway. In the ethylene-insensitive mutants etr1-3 and ein2-1, cytokinin treatment resulted in hypocotyl lengths comparable to those of wild-type seedlings treated with both Ag⁺ and BA. A similar phenotypical response to cytokinin was observed when auxin transport was blocked by -naphthylphthalamic acid (NPA). Applied cytokinin largely restored cell elongation in the basal and middle parts of the hypocotyls of NPA-treated seedlings and at the same time abolished the NPA-induced decrease in indole-3-acetic acid levels. Our data support the hypothesis that, in the light, cytokinins interact with the ethylene-signalling pathway and conditionally up-regulate ethylene and auxin synthesis.     

Characteristic and expression analysis of a metallothionein gene, OsMT2b, down-regulated by cytokinin suggests functions in root development and seed embryo germination of rice

Metallothioneins (MTs) are low molecular mass and cysteine-rich metal-binding proteins known to be mainly involved in maintaining metal homeostasis and stress responses. But, their functions in higher plant development are scarcely studied. Here, we characterized rice (Oryza sativa) METALLOTHIONEIN2b (OsMT2b) molecularly and found that its expression was down-regulated by cytokinins. OsMT2b was preferentially expressed in rice immature panicles, scutellum of germinating embryos, and primordium of lateral roots. In contrast with wild-type plants, OsMT2b-RNA interference (RNAi) transgenic plants had serious handicap in plant growth and root formation, whereas OsMT2b-overexpressing transformants were dwarfed and presented more adventitious roots and big lateral roots. The increased cytokinin levels in RNAi plants and decreased cytokinin levels in overexpressing plants were confirmed by high-performance liquid chromatography quantitative analysis in the roots of wild-type and transgenic plants. In RNAi plants, localization of isopentenyladenosine, a kind of endogenous cytokinin, in roots and germinating embryos expanded to the whole tissues, whereas in overexpressing plants, the isopentenyladenosine signals were very faint in the vascular tissues of roots and scutellum cells of germinating embryos. In vitro culture of embryos could largely resume the reduced germination frequency in RNAi plants but had no obvious change in overexpressing plants. Taken together, these results indicate a possible feedback regulation mechanism of OsMT2b to the level of endogenous cytokinins that is involved in root development and seed embryo germination of rice.