Spatio-temporal changes in endogenous abscisic acid contents during etiolated growth and photomorphogenesis in tomato seedlings

The role of abscisic acid (ABA) during early development was investigated in tomato seedlings. The endogenous content of ABA in particular organs was analyzed in seedlings grown in the dark and under blue light. Our results showed that in dark-grown seedlings, the ABA accumulation was maximal in the cotyledons and elongation zone of hypocotyl, whereas under blue-light, the ABA content was distinctly reduced. Our data are consistent with the conclusion that ABA promotes the growth of etiolated seedlings and the results suggest that ABA plays an inhibitory role in de-etiolation and photomorphogenesis in tomato.     

Genetic dissection of blue-light sensing in tomato using mutants deficient in cryptochrome 1 and phytochromes A, B1 and B2

Several novel allelic groups of tomato (Solanum lycopersicum L.) mutants with impaired photomorphogenesis have been identified after gamma-ray mutagenesis of phyA phyB1 double-mutant seed. Recessive mutants in one allelic group are characterized by retarded hook opening, increased hypocotyl elongation and reduced hypocotyl chlorophyll content under white light (WL). These mutants showed a specific impairment in response to blue light (BL) resulting from lesions in the gene encoding the BL receptor cryptochrome 1 (cry1). Phytochrome A and cry1 are identified as the major photoreceptors mediating BL-induced de-etiolation in tomato, and act under low and high irradiances, respectively. Phytochromes B1 and B2 also contribute to BL sensing, and the relative contribution of each of these four photoreceptors differs according to the light conditions and the specific process examined. Development of the phyA phyB1 phyB2 cry1 quadruple mutant under WL is severely impaired, and seedlings die before flowering. The quadruple mutant is essentially blind to BL, but experiments employing simultaneous irradiation with BL and red light suggest that an additional non-phytochrome photoreceptor may be active under short daily BL exposures. In addition to effects on de-etiolation, cry1 is active in older, WL-grown plants, and influences stem elongation, apical dominance, and the chlorophyll content of leaves and fruit. These results provide the first mutant-based characterization of cry1 in a plant species other than Arabidopsis.     

Changes in Concentrations of Cytokinins (CKs) in Root and Axillary Bud Tissue of Miniature Rose Suggest that Local CK Biosynthesis and Zeatin-Type CKs Play Important Roles in Axillary Bud Growth

Involvement of cytokinins (CKs) in axillary bud growth of miniature rose was studied. Variation in root formation and axillary bud growth was induced by two indole 3-butyric acid (IBA) pretreatments in two cutting sizes. At six physiological developmental stages around the onset of axillary bud growth, concentrations of CKs were determined in both root and axillary bud tissue by liquid chromatography combined with electrospray tandem mass spectrometry (LC-ESP-MS/MS). Chronological early onset of axillary bud growth occurred in long cuttings pretreated at low IBA concentration, whereas physiological early root formation was associated with long cuttings and high IBA concentration. The CKs zeatin (Z), isopentenyl adenine (iP), zeatin riboside (ZR), dihydrozeatin riboside (DHZR), isopentenyl adenosine (iPA), zeatin O-glucoside (ZOG), zeatin riboside O-glucoside (ZROG), zeatin riboside 5′-monophosphate (ZRMP), and isopentenyl adenosine 5′-monophosphate (iPAMP) were detected. Concentrations of CKs in axillary bud tissue far exceeded those in root tissue. Indole 3-butyric acid pretreatment influenced the concentration of CKs in axillary bud tissue more than did cutting size, whereas pretreatments only slightly affected CKs in root tissue. The dominant CKs found were iPAMP and ZR. An early and large increase in iPAMP indicated rapid CK biosynthesis in rootless cuttings, suggesting that green parts, including the axillary bud, can synthesize CKs. At the onset of axillary bud growth an increase in concentration of Z, ZR, ZRMP, ZOG, and ZROG was largely coincident with a decrease in iPAMP, iPA, iP, and DHZR. After the onset of axillary bud growth, CK content largely decreased. These results strongly indicate a positive role for CKs in axillary bud growth, and presumably ZRMP, ZR, and Z are active in miniature rose.     

Large-scale Identification and Time-course Quantification of Ubiquitylation Events During Maize Seedling De-etiolation

The ubiquitin system is crucial for the development and fitness of higher plants. De-etiolation, during which green plants initiate photomorphogenesis and establish autotrophy, is a dramatic and complicated process that is tightly regulated by a massive number of ubiquitylation/de-ubiquitylation events. Here we present site-specific quantitative proteomic data for the ubiquitylomes of de-etiolating seedling leaves of Zea mays L. (exposed to light for 1, 6, or 12 h) achieved through immunoprecipitation-based high-resolution mass spectrometry (MS). Through the integrated analysis of multiple ubiquitylomes, we identified and quantified 1926 unique ubiquitylation sites corresponding to 1053 proteins. We analyzed these sites and found five potential ubiquitylation motifs, KA, AXK, KXG, AK, and TK. Time-course studies revealed that the ubiquitylation levels of 214 sites corresponding to 173 proteins were highly correlated across two replicate MS experiments, and significant alterations in the ubiquitylation levels of 78 sites (fold change >1.5) were detected after de-etiolation for 12 h. The majority of the ubiquitylated sites we identified corresponded to substrates involved in protein and DNA metabolism, such as ribosomes and histones. Meanwhile, multiple ubiquitylation sites were detected in proteins whose functions reflect the major physiological changes that occur during plant de-etiolation, such as hormone synthesis/signaling proteins, key C4 photosynthetic enzymes, and light signaling proteins. This study on the ubiquitylome of the maize seedling leaf is the first attempt ever to study the ubiquitylome of a C4 plant and provides the proteomic basis for elucidating the role of ubiquitylation during plant de-etiolation.     

Calcium and calcium receptor CAS promote Arabidopsis thaliana de-etiolation

As a second messenger, the free cytosolic calcium ion (Ca(2+)) plays important roles in many biochemical and physiological processes including photosynthesis in plants. In this study, we investigated morphological changes, chlorophyll accumulation and chloroplast development during early photomorphogenesis in etiolated seedlings of both Arabidopsis thaliana wild type (WT) and those with the antisense of CAS, a calcium sensor (CASas). Seedlings were grown at high, medium and low Ca(2+) concentrations to identify the roles of Ca(2+) and CAS in de-etiolation and chloroplast development. The results demonstrated that Ca(2+) and CAS are correlated with de-etiolation of A. thaliana after light exposure. High Ca(2+) significantly increased chlorophyll content and improved chloroplast development in both A. thaliana WT and CASas etiolated seedlings during de-etiolation. The analysis by western blot and real-time fluorescent quantitative polymerase chain reaction indicated that the expression levels of CAS mRNA and protein were upregulated by white light and external Ca(2+) significantly. Etiolated CASas plants showed much lower chlorophyll content and delay of chloroplast development as compared with WT plants, indicating that CAS functions in de-etiolation. All together, we concluded that the de-etiolation in A. thaliana was promoted by the high Ca(2+) concentration and CAS expression to a certain extent.     

Gibberellins repress photomorphogenesis in darkness

Plants undergo two different developmental programs depending on whether they are growing in darkness (skotomorphogenesis) or in the presence of light (photomorphogenesis). It has been proposed that the latter is the default pathway followed by many plants after germination and before the seedling emerges from soil. The transition between the two pathways is tightly regulated. The conserved COP1-based complex is central in the light-dependent repression of photomorphogenesis in darkness. Besides this control, hormones such as brassinosteroids (BRs), cytokinins, auxins, or ethylene also have been shown to regulate, to different extents, this developmental switch. In the present work, we show that the hormone gibberellin (GA) widely participates in this regulation. Studies from Arabidopsis show that both chemical and genetic reductions of endogenous GA levels partially derepress photomorphogenesis in darkness. This is based both on morphological phenotypes, such as hypocotyl elongation and hook and cotyledon opening, and on molecular phenotypes, such as misregulation of the light-controlled genes CAB2 and RbcS. Genetic studies indicate that the GA signaling elements GAI and RGA participate in these responses. Our results also suggest that GA regulation of this response partially depends on BRs. This regulation seems to be conserved across species because lowering endogenous GA levels in pea (Pisum sativum) induces full de-etiolation in darkness, which is not reverted by BR application. Our results, therefore, attribute an important role for GAs in the establishment of etiolated growth and in repression of photomorphogenesis.     

Endogenous cytokinin dynamics in micropropagated tulips during bulb formation process influenced by TDZ and iP pretreatment

There are substantial variations in bulbing (bulb formation) efficiency among micropropagated tulip cultivars. The mechanisms involved are poorly understood, but presumably involve cytokinins (CKs) for several reasons. Therefore, we explored CK profiles and dynamics in ‘Blue Parrot’ and ‘Prominence’ cultivars (which have low and high bulbing efficiency, respectively) during the in vitro propagation stages: the last shoot multiplication subculture extended to 14 weeks (S1–S2), the shoot cooling at 5 °C for induction of bulb formation (S3–S4) and the bulb growth initiation after the end of cooling (S5–S6). The CK thidiazuron (TDZ) is routinely used in tulip micropropagation at the shoot multiplication stage, but replacing it with isopentenyladenine (iP) during the last multiplication subculture substantially changed CK dynamics in later stages, and significantly increased bulb formation rates in both cultivars. Generally, the most abundant CKs in both cultivars were the isoprenoid CK types, trans-zeatin (tZ), iP, cis-zeatin and dihydrozeatin. However, ‘Prominence’ shoots had much lower cis- to trans-Z-type CK ratios than ‘Blue Parrot’ shoots, and generally higher levels of physiologically active CKs (free bases tZ, iP and their ribosides) until the last phase of bulb formation, S6 (bulb growth initiation, i.e. swelling of shoot bases), 6 weeks after the end of cold treatment. In this phase total active CK and O-glucoside contents sharply declined in ‘Prominence’ shoots, but not in ‘Blue Parrot’ shoots pretreated with iP. In contrast, the low bulbing ability observed in ‘Prominence’ shoots pretreated with TDZ and ‘Blue Parrot’ shoots pretreated with either TDZ or iP was associated with a gradual rise in active CK and O-glucoside contents after the end of cooling. The results suggest that low bulbing efficiency may be related to down-regulation of tZ biosynthesis, and high bulbing efficiency to a transient increase in active CK forms (mainly tZs) in response to cold treatment during the bulb induction phase, S4 (at the end of cold treatment), followed by a rapid decrease during bulb formation, S6 (6 weeks after the end of cooling).     

Arabidopsis light-dependent protochlorophyllide oxidoreductase A (PORA) is essential for normal plant growth and development

During skotomorphogenesis in angiosperms, NADPH:protochlorophyllide oxidoreductase (POR) forms an aggregate of photolabile NADPH-POR-protochlorophyllide (Pchlide) ternary complexes localized to the prolamellar bodies within etioplasts. During photomorphogenesis, POR catalyzes the light-dependent reduction of Pchlide a to chlorophyllide (Chlide) a, which is subsequently converted to chlorophyll (Chl). In Arabidopsis there are three structurally related POR genes, denoted PORA, PORB and PORC. The PORA and PORB proteins accumulate during skotomorphogenesis. During illumination, PORA is only transiently expressed, whereas PORB and PORC persist and are responsible for bulk Chl synthesis throughout plant development. Here we have tested whether PORA is important for skotomorphogenesis by assisting in etioplast development, and normal photomorphogenic development. Using reverse genetic approaches, we have identified the porA-1 null mutant, which contains an insertion of the maize Dissociation transposable element in the PORA gene. Additionally, we have characterized PORA RNAi lines. The porA-1 and PORA RNAi lines display severe photoautotrophic growth defects, which can be partially rescued on sucrose-supplemented growth media. Elimination of PORA during skotomorphogenesis results in reductions in the volume and frequency of prolamellar bodies, and in photoactive Pchlide conversion. The porA-1 mutant characterization thus establishes a quantitative requirement for PORA in etioplast development by demonstrating significant membrane ultrastructural and biochemical defects, in addition to suggesting PORA-specific functions in photomorphogenesis and plant development.     

Physiological interactions of phytochromes A, B1 and B2 in the control of development in tomato

The role of phytochrome B2 (phyB2) in the control of photomorphogenesis in tomato (Solanum lycopersicum L.) has been investigated using recently isolated mutants carrying lesions in the PHYB2 gene. The physiological interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phyB2 have also been explored, using an isogenic series of all possible mutant combinations and several different phenotypic characteristics. The loss of phyB2 had a negligible effect on the development of white-light-grown wild-type or phyA-deficient plants, but substantially enhanced the elongated pale phenotype of the phyB1 mutant. This redundancy was also seen in the control of de-etiolation under continuous red light (R), where the loss of phyB2 had no detectable effect in the presence of phyB1. Under continuous R, phyA action was largely independent of phyB1 and phyB2 in terms of the control of hypocotyl elongation, but antagonized the effects of phyB1 in the control of anthocyanin synthesis, indicating that photoreceptors may interact differently to control different traits. Irradiance response curves for anthocyanin synthesis revealed that phyB1 and phyB2 together mediate all the detectable response to high-irradiance R, and, surprisingly, that the phyA-dependent low-irradiance component is also strongly reduced in the phyB1 phyB2 double mutant. This is not associated with a reduction in phyA protein content or responsiveness to continuous far-red light (FR), suggesting that phyB1 and phyB2 specifically influence phyA activity under low-irradiance R. Finally, the phyA phyB1 phyB2 triple mutant showed strong residual responsiveness to supplementary daytime FR, indicating that at least one of the two remaining phytochromes plays a significant role in tomato photomorphogenesis.     

The role of COP1 in light control of Arabidopsis seedling development

The Arabidopsis seedling can follow two contrasting developmental programmes, photomorphogenesis in light and skotomorphogenesis in darkness. CONSTITUTIVE PHOTOMORPHOGENIC I (COP1) is an essential regulatory gene required for repression of seedling photomorphogenic development in darkness. Recent mutational and overexpression analyses of the COP1 gene suggest a central role for COP1 in light control of seedling development and point to functional implications of its structural domains. Cell biological studies of COP1 have provided a clue to how light regulates the repressive activity of COP1 and thus seedling development.     

How does exogenously applied cytokinin type affect growth and endogenous cytokinins in micropropagated Merwilla plumbea?

Merwilla plumbea (Lindl.) Speta is a popular and highly sought after South African medicinal plant with diverse therapeutic values. Using Ultra performance liquid chromatography (UPLC), the effect of five cytokinins (CKs) [either isoprenoid = N ⁶-isopentenyladenine (iP) or aromatic = benzyladenine, meta-topolin (mT), meta-topolin riboside (mTR), and 6-(3-methoxybenzylamino)-9-tetrahydropyran-2-ylpurine] MemTTHP on growth and level of endogenous CKs during micropropagation and acclimatization stages was evaluated. Aromatic CK (mT/mTR) elicited the highest shoot proliferation (7–8 shoots per explant) during in vitro culture. Following acclimatization, iP-treated and the control plants were healthier with longer leaves, roots and higher fresh weight when compared to aromatic CKs. A total of 37 (22 isoprenoid and 15 aromatic) CK variants were quantified in both in vitro and acclimatized plants. Based on their metabolic function, these were grouped into five types including free bases, ribosides, ribotides, O- and 9-glucosides. In addition to enhancing our understanding of the hormone physiology in M. plumbea, the current findings are discussed in line with the effect of the exogenously applied CK on the observed differences in growth before and after the important stage of acclimatization. The observed dynamics in endogenous CK provide an avenue to manipulate in vitro growth and development of investigated species.     

Large-scale Proteomic and Phosphoproteomic Analyses of Maize Seedling Leaves During De-etiolation

De-etiolation consists of a series of developmental and physiological changes that a plant undergoes in response to light. During this process light, an important environmental signal, triggers the inhibition of mesocotyl elongation and the production of photosynthetically active chloroplasts, and etiolated leaves transition from the “sink” stage to the “source” stage. De-etiolation has been extensively studied in maize (Zea mays L.). However, little is known about how this transition is regulated. In this study, we described a quantitative proteomic and phosphoproteomic atlas of the de-etiolation process in maize. We identified 16,420 proteins in proteome, among which 14,168 proteins were quantified. In addition, 8746 phosphorylation sites within 3110 proteins were identified. From the combined proteomic and phosphoproteomic data, we identified a total of 17,436 proteins. Only 7.0% (998/14,168) of proteins significantly changed in abundance during de-etiolation. In contrast, 26.6% of phosphorylated proteins exhibited significant changes in phosphorylation level; these included proteins involved in gene expression and homeostatic pathways and rate-limiting enzymes involved in photosynthetic light and carbon reactions. Based on phosphoproteomic analysis, 34.0% (1057/3110) of phosphorylated proteins identified in this study contained more than 2 phosphorylation sites, and 37 proteins contained more than 16 phosphorylation sites, indicating that multi-phosphorylation is ubiquitous during the de-etiolation process. Our results suggest that plants might preferentially regulate the level of posttranslational modifications (PTMs) rather than protein abundance for adapting to changing environments. The study of PTMs could thus better reveal the regulation of de-etiolation.     

Endogenous Abscisic Acid Promotes Hypocotyl Growth and Affects Endoreduplication during Dark-Induced Growth in Tomato (Solanum lycopersicum L.)

Dark-induced growth (skotomorphogenesis) is primarily characterized by rapid elongation of the hypocotyl. We have studied the role of abscisic acid (ABA) during the development of young tomato (Solanum lycopersicum L.) seedlings. We observed that ABA deficiency caused a reduction in hypocotyl growth at the level of cell elongation and that the growth in ABA-deficient plants could be improved by treatment with exogenous ABA, through which the plants show a concentration dependent response. In addition, ABA accumulated in dark-grown tomato seedlings that grew rapidly, whereas seedlings grown under blue light exhibited low growth rates and accumulated less ABA. We demonstrated that ABA promotes DNA endoreduplication by enhancing the expression of the genes encoding inhibitors of cyclin-dependent kinases SlKRP1 and SlKRP3 and by reducing cytokinin levels. These data were supported by the expression analysis of the genes which encode enzymes involved in ABA and CK metabolism. Our results show that ABA is essential for the process of hypocotyl elongation and that appropriate control of the endogenous level of ABA is required in order to drive the growth of etiolated seedlings.     

Photomorphogenic responses to UV radiation IV: a comparative study of UVB effects on growth and pigment accumulation in etiolated and de-etiolated hypocotyls of wild-type and aurea mutant of tomato Lycopersicon esculentum Mill)

ABSTRACT

The question of how de-etiolation of tomato seedling under continuous monochromatic yellow light exerts an influence on UV radiation-induced responses has been studied. Hypocotyl extension and the accumulation of anthocyanins and UV-absorbing compounds was compared in the aurea mutant of tomato and its isogenic wild type. The data of the present paper provide evidence that, during de-etiolation of tomato seedlings, yellow light exerts its effects over seedlings responsiveness to subsequent UV irradiation through several mechanisms: 1) a significant enhancement of shorter UVB wavelength efficiency in both the genotypes; 2) the abolition of UVA -blue light-induced accumulation of UV-absorbing compounds that does not involve pnyA; 3) the disappearance of UVA-blue light-induced hypocotyl growth inhibition that does not involve phyA; 4) higher anthocyanin accumulation rate in response to UV radiation mediated by phyA. Yellow light-induced growth inhibition and accumulation of UV-absorbing compound both mediated by phyA and present only in wild type tomato, appear to be completely separate from the action of UV radiation on the same responses.     

Role of Phytohormones and Light in De-etiolation

Russian Journal of Plant Physiology - De-etiolation or transition from etiolated growth (skotomorphogenesis) to photomorphogenesis is one of the most intriguing and intricate stages of plant...