Identification of Differentially Expressed Proteins and Phosphorylated Proteins in Rice Seedlings in Response to Strigolactone Treatment

Strigolactones (SLs) are recently identified plant hormones that inhibit shoot branching and control various aspects of plant growth, development and interaction with parasites. Previous studies have shown that plant D10 protein is a carotenoid cleavage dioxygenase that functions in SL biosynthesis. In this work, we used an allelic SL-deficient d10 mutant XJC of rice (Oryza sativa L. spp. indica) to investigate proteins that were responsive to SL treatment. When grown in darkness, d10 mutant seedlings exhibited elongated mesocotyl that could be rescued by exogenous application of SLs. Soluble protein extracts were prepared from d10 mutant seedlings grown in darkness in the presence of GR24, a synthetic SL analog. Soluble proteins were separated on two-dimensional gels and subjected to proteomic analysis. Proteins that were expressed differentially and phosphoproteins whose phosphorylation status changed in response to GR24 treatment were identified. Eight proteins were found to be induced or down-regulated by GR24, and a different set of 8 phosphoproteins were shown to change their phosphorylation intensities in the dark-grown d10 seedlings in response to GR24 treatment. Analysis of these proteins revealed that they are important enzymes of the carbohydrate and amino acid metabolic pathways and key components of the cellular energy generation machinery. These proteins may represent potential targets of the SL signaling pathway. This study provides new insight into the complex and negative regulatory mechanism by which SLs control shoot branching and plant development.     

Chemical screening of novel strigolactone agonists that specifically interact with DWARF14 protein

Strigolactones (SLs) are a class of plant hormones that regulate shoot branching as well as being known as root-derived signals for parasitic and symbiotic interactions. The physical interaction between SLs and the DWARF14 (D14) receptor family can be examined by differential scanning fluorimetry (DSF) that monitors the changes in protein melting temperature (Tm). The Tm of D14 is lowered by bioactive SLs in DSF analysis. In this report, we screened the compounds that lower the Tm of Arabidopsis D14 (AtD14) as potential candidates for SL agonists using DSF analysis. Subsequent physiological analyzes revealed that 113D10 acts as a novel SL agonist in a D14-dependent manner. Intriguingly, 113D10 has a chemical structure different from natural SLs in that it does not possess an enol ether bond that connects to a methylbutenolide moiety. Moreover, 113D10 does not stimulate seed germination of root parasitic plants. Accordingly, 113D10 can be a useful tool for SL studies and agricultural applications.     

Strigolactones interact with ethylene and auxin in regulating root-hair elongation in Arabidopsis

Strigolactones (SLs) or derivatives thereof have been identified as phytohormones, and shown to act as long-distance shoot-branching inhibitors. In Arabidopsis roots, SLs have been suggested to have a positive effect on root-hair (RH) elongation, mediated via the MAX2 F-box. Two other phytohormones, auxin and ethylene, have been shown to have positive effects on RH elongation. Hence, in the present work, Arabidopsis RH elongation was used as a bioassay to determine epistatic relations between SLs, auxin, and ethylene. Analysis of the effect of hormonal treatments on RH elongation in the wild type and hormone-signalling mutants suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs, whereas the effect of SLs on RH elongation requires ethylene synthesis. SL signalling was not needed for the auxin response, whereas auxin signalling was not necessary, but enhanced RH response to SLs, suggesting that the SL and auxin hormonal pathways converge for regulation of RH elongation. The ethylene pathway requirement for the RH response to SLs suggests that ethylene forms a cross-talk junction between the SL and auxin pathways.     

Increased molecular mass of hemicellulosic polysaccharides is involved in growth inhibition of maize coleoptiles and mesocotyls under hypergravity conditions.

Elongation growth of dark grown maize (Zea mays L cv. Cross Bantam T51) coleoptiles and mesocotyls was suppressed by hypergravity at 30 g and above. Acceleration at 300 g significantly decreased the mechanical extensibility of cell walls of both organs. Hypergravity increased the amounts of hemicellulose and cellulose per unit length in mesocotyl walls, but not in coleoptile walls. The weight average molecular masses of hemicellulosic polysaccharides were also increased by hypergravity in both organs. On the other hand, the activities of beta-glucanases extracted from coleoptile and mesocotyl cell walls were decreased by hypergravity. These results suggest that the decreased activities of beta-glucanases by hypergravity cause an increase in the molecular mass of hemicellulosic polysaccharides of both organs. The upshift of molecular mass of hemicellulosic polysaccharides as well as the thickening of cell walls under hypergravity conditions seems to be involved in making the cell wall mechanically rigid, thereby inhibiting elongation growth of maize coleoptiles and mesocotyls.     

Effects of abscisic acid and its related compounds on rice seedling growth

Rice seedlings with the mesocotyl and coleoptile (the undeveloped leaves enclosed in the coleoptile) are here referred to as MC type seedlings and are considered to be suitable for deep sowing. We investigated the effects of abscisic acid (ABA) and several of its related compounds on the occurrence of MC type seedlings and on rice mesocotyl growth. Rice (Oryza sativa L. cv. JC 91) seedlings were grown on 0.8% agar medium in the presence or absence of various kinds of ABAs under aseptic conditions at 30 °C in the dark for 14 days. The activity of the R isomer of ABA (R-ABA) was slightly less than that of the naturally occurring S form (S-ABA) concerning the occurrence of MC type rice seedlings and the growth of the rice mesocotyl. In addition, the racemate of R-and S-ABA (RS-ABA) is less effective than R-ABA and S-ABA alone.Trans-ABA had no activity in relation to both percent occurrence of MC type seedlings and mesocotyl growth. The results of the present study suggest that the occurrence of MC type rice seedlings and the growth of rice mesocotyls were closely related to structure-activity relationships with analogs of ABA.     

Characterization of a new allelic mutant of DWARF3 in rice and analysing its function and stability in the presence of strigolactone

Strigolactones (SLs) are important intrinsic growth regulators that control plant architecture by coordinating shoot and root development. Recent studies demonstrate that SL signals act via targeting the degradation protein DWARF53 (D53) family of chaperonin-like proteins. This process requires DWARF14 (D14) as strigolactones signal receptor and DWARF3 (D3) forming Skp-Cullin-F-box (SCF) complex as ubiquitin E3 ligase. Although the interactions of these signal components can be expected, where and how the SLs signalling occur within cells in a tissue-specific manner is still uncertain. In this study, we characterize a rice high-tillering dwarf mutant, ext.-M1B, displaying resistance to synthetic strigolactone mixture rac-GR24. Through genetic analysis, we find that ext.-M1B is a new allelic mutant of D3 with a nucleotide mutation resulting in a truncated protein of wide-type D3. We demonstrate that the mutation affects neither gene expression level nor the protein sub-cellular localization, whereas it disrupts the perception of SLs signal in ext.-M1B mutant. Moreover, we find that overexpression of D3 in wild type background causes no significant phenotype, but suppression of D3 by RNA interfering results in a clear phenocopy of SL mutants. By expressing fluorescent D3 fusion protein in rice, we first show that D3 is stable consistently in the nucleus with or without strigolactone treatment. Taken together, our data indicates that D3 encoding an F-box protein in nucleus, as a stable signal component response to strigolactone regulating rice shoot architecture.     

Response of actin microfilaments during phytochrome-controlled growth of maize seedlings

Summary In seedlings of maize (Zea mays L. cv. Percival), growth is controlled by the plant photoreceptor phytochrome. Whereas coleoptile growth is promoted by continuous far-red light, a dramatic block of mesocotyl elongation is observed. The response of the coleoptile is based entirely upon light-induced stimulation of cell elongation, whereas the response of the mesocotyl involves light-induced inhibition of cell elongation. The light response of actin microfilaments was followed over time in the epidermis by staining with fluorescence-labelled phalloidin. In contrast to the underlying tissue, epidermal cells are characterized by dense longitudinal bundles of microfilaments. These bundles become loosened during phases of rapid elongation (between 2–3 days in irradiated coleoptiles, between 5–6 days in dark-grown coleoptiles). The condensed bundles re-form when growth gradually ceases. The response of actin to light is fast. If etiolated mesocotyls are transferred to far-red light, condensation of microfilaments can be clearly seen 1 h after the onset of stimulation together with an almost complete block of mesocotyl elongation. The observations are discussed in relation to a possible role of actin microfilaments in the signal-dependent control of cell elongation.     

Phenylalanine Ammonia-Lyase Activity and Anthocyanin Accumulation in Wounded Maize Mesocotyls

Activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5) and anthocyanin accumulation were determined in wounded maize (Zea mays L.) mesocotyls. Mesocotyls were wounded with aluminum oxide and were placed in a 15 h light: 9 h dark photoperiod or in the dark. Extractable enzyme activity increased in response to wounding in the photoperiod but not in the dark. Anthocyanin accumulation in mesocotyls placed in the photoperiod decreased in response to wounding. The results are discussed with reference to phenylalanine ammonia-lyase activity in mesocotyl tissue wounded or inoculated with fungal pathogens.     

The role of strigolactones in root development

Strigolactones (SLs) and their derivatives were recently defined as novel phytohormones that orchestrate shoot and root growth. Levels of SLs, which are produced mainly by plant roots, increase under low nitrogen and phosphate levels to regulate plant responses. Here, we summarize recent work on SL biology by describing their role in the regulation of root development and hormonal crosstalk during root deve-lopment. SLs promote the elongation of seminal/primary roots and adventitious roots (ARs) and they repress lateral root formation. In addition, auxin signaling acts downstream of SLs. AR formation is positively or negatively regulated by SLs depending largely on the plant species and experimental conditions. The relationship between SLs and auxin during AR formation appears to be complex. Most notably, this hormonal response is a key adaption that radically alters rice root architecture in response to nitrogen- and phosphate-deficient conditions.     

Morphological and anatomical effects of abscisic acid (ABA) and fluridone (FLU) on the growth of rice mesocotyls

It has been reported that abscisic acid (ABA) promotes the growth ofrice mesocotyls. We investigated the effects of ABA and fluridone (FLU), aninhibitor of ABA biosynthesis, on the morphological and anatomicalcharacteristics of the mesocotyl of dark-grown rice seedlings. By 5 days aftersowing (DAS), the mesocotyl of control seedlings had reached their a maximumlength of 50.0 mm. In ABA treated seedlings, the mesocotylcontinued growing until 12 DAS, reaching a length of 152.6 mm.Mesocotyls treated with FLU elongated until 4DAS attaining a final length ofonly 10.5 mm. The data indicated that the greater length ofmesocotyls in seedlings treated with ABA was mainly due to their longer growingperiod. An anatomical study showed that the cell division activity of themesocotyl localised near the coleoptilar node and that ABA prolonged the celldivision activity of the meristem. The results suggested that ABA regulates thegrowth of rice mesocotyls by controlling the activity of the meristem localisednear the coleoptilar node.     

Changes in cell wall composition of rice mesocotyls in relation to growth and treatment with 4-ethoxy-1-(p-tolyl)-s-triazine-2,6(1H, 3H)-dione

A synthetic substance, 4-ethoxy-l-(p-tolyl)-s-triazine-2,6(1H,3H)-dione [TA] dramatically promoted mesocotyl growth in dark-grownrice (Oryza sativa L. cv. Nato) seedlings, the optimal concentrationbeing 0.1–0.2 mM. Changes in the cell wall compositionof the rice mesocotyls were examined in relation to growth andtreatment with 0.1 mM TA. The amount of the cell wall increasedduring the elongation in control and treated mesocotyls. Particularly,TA caused a large increase in the amount of the cell wall permesocotyl but a decrease per unit length of mesocotyl. Hydrolysisof the cell wall with trifluoroacetic acid liberated xylose,glucose, arabinose, galactose, and trace amounts of rhamnose,fucose and mannose. An increase in the relative amount of xyloseand a decrease in that of glucose in the noncellulosic fractionduring growth were found in control and treated mesocotyl walls.On the 2nd day after planting when the mesocotyl emerged, TAsignificantly affected the cell wall composition; TA decreasedthe relative amount of -cellulose in the wall, and caused anincrease in the relative amount of glucose and decreases inthose of xylose and arabinose in the noncellulosic fraction. ¹This paper is Part 7 in the series "Plant Growth-regulatingActivities of Isourea Derivatives and Related Compounds." (Received March 18, 1980; )     

独脚金内酯调控水稻分蘖的研究进展

水稻(Oryza sativa)作为世界上最主要的粮食作物之一, 对其主要农艺性状调控机理的研究具有重要意义。分蘖是水稻生长发育过程中一种特殊的分枝, 它不仅是与水稻产量密切相关的重要农艺性状, 也是揭示高等植物侧枝生长发育机制的理想模型。独脚金内酯(strigolactone, SL)是一类新型植物激素, 能够抑制植物分枝的生长发育。近年来, 关于SL合成与信号在调控水稻分蘖方面的研究取得了重要进展, 但对其信号转导的下游组分的研究还相对匮乏。该文综述了SL合成途径、信号途径及下游靶基因调控水稻分蘖的研究进展, 并与在拟南芥(Arabidopsis thaliana)、豌豆(Pisum sativum)和矮牵牛(Petunia hybrida)中的研究进行了比较, 同时还对如何挖掘SL途径的新组分进行了讨论。     

Effect of auxin on mesocotyl elongation of dark-grown maize under different seeding depths

In order to elucidate the physiological mechanism of maize mesocotyl elongation induced by auxin under different seeding depths, seeds of five maize inbred lines, including 3681-4 line tolerant to deep seeding, were treated with IAA and triiodobenzoic acid (TIBA) under seeding depths of 20 or 2 cm. Under deep seeding conditions, maize mesocotyls grew by 1.5–2.0 times faster than under shallow seeding conditions. IAA (10⁻⁶ to 10⁻⁴ M) applied to roots stimulated mesocotyl elongation only of 3681-4 line and only under deep seeding conditions. TIBA (10⁻⁵ and 10⁻⁴ M) applied to roots inhibited mesocotyl elongation in all lines, but only 3681-4 was sensitive to 10⁻⁶ M TIBA. IAA promoted only cell elongation, and TIBA inhibited both cell elongation and cell division. After IAA and TIBA treatments, endogenous IAA content changed in parallel with the mesocotyl growth rate under different seeding depths. Furthermore, ABP1 gene expression changed in parallel with the mesocotyl growth rate under deep seeding conditions. Therefore, deep seeding tolerance of 3681-4 line was achieved due to auxin-regulated rapid mesocotyl elongation.     

Strigolactones are required for nitric oxide to induce root elongation in response to nitrogen and phosphate deficiencies in rice

The response of the root system architecture to nutrient deficiencies is critical for sustainable agriculture. Nitric oxide (NO) is considered a key regulator of root growth, although the mechanisms remain unknown. Phenotypic, cellular and genetic analyses were undertaken in rice to explore the role of NO in regulating root growth and strigolactone (SL) signalling under nitrogen‐deficient and phosphate‐deficient conditions (LN and LP). LN‐induced and LP‐induced seminal root elongation paralleled NO production in root tips. NO played an important role in a shared pathway of LN‐induced and LP‐induced root elongation via increased meristem activity. Interestingly, no responses of root elongation were observed in SL d mutants compared with wild‐type plants, although similar NO accumulation was induced by sodium nitroprusside (SNP) application. Application of abamine (the SL inhibitor) reduced seminal root length and pCYCB1;1::GUS expression induced by SNP application in wild type; furthermore, comparison with wild type showed lower SL‐signalling genes in nia2 mutants under control and LN treatments and similar under SNP application. Western blot analysis revealed that NO, similar to SL, triggered proteasome‐mediated degradation of D53 protein levels. Therefore, we presented a novel signalling pathway in which NO‐activated seminal root elongation under LN and LP conditions, with the involvement of SLs.     

The Physcomitrium (Physcomitrella) patens PpKAI2L receptors for strigolactones and related compounds function via MAX2-dependent and -independent pathways

In angiosperms, the α/β hydrolase DWARF14 (D14), along with the F-box protein MORE AXILLARY GROWTH2 (MAX2), perceives strigolactones (SL) to regulate developmental processes. The key SL biosynthetic enzyme CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) is present in the moss Physcomitrium patens, and PpCCD8-derived compounds regulate moss extension. The PpMAX2 homolog is not involved in the SL response, but 13 PpKAI2LIKE (PpKAI2L) genes homologous to the D14 ancestral paralog KARRIKIN INSENSITIVE2 (KAI2) encode candidate SL receptors. In Arabidopsis thaliana, AtKAI2 perceives karrikins and the elusive endogenous KAI2-Ligand (KL). Here, germination assays of the parasitic plant Phelipanche ramosa suggested that PpCCD8-derived compounds are likely noncanonical SLs. (+)-GR24 SL analog is a good mimic for PpCCD8-derived compounds in P. patens, while the effects of its enantiomer (−)-GR24, a KL mimic in angiosperms, are minimal. Interaction and binding assays of seven PpKAI2L proteins pointed to the stereoselectivity toward (−)-GR24 for a single clade of PpKAI2L (eu-KAI2). Enzyme assays highlighted the peculiar behavior of PpKAI2L-H. Phenotypic characterization of Ppkai2l mutants showed that eu-KAI2 genes are not involved in the perception of PpCCD8-derived compounds but act in a PpMAX2-dependent pathway. In contrast, mutations in PpKAI2L-G, and -J genes abolished the response to the (+)-GR24 enantiomer, suggesting that PpKAI2L-G, and -J proteins are receptors for moss SLs.

The study of moss PpKAI2L receptors for strigolactones and related compounds highlights MORE AXILLARY GROWTH2-dependent and -independent pathways for the perception of these compounds.