Phenotypic and Genetic Analysis of det2, a New Mutant That Affects Light-Regulated Seedling Development in Arabidopsis

The greening phenotypes produced by recessive mutations in a gene designated de-etiolated-2 (DET2) are described. Recessive mutations in the DET2 gene uncouple light signals from a number of light-dependent processes. det2 mutations result in dark-grown Arabidopsis thaliana seedlings with many characteristics of light-grown plants, including hypocotyl growth inhibition, cotyledon expansion, primary leaf initiation, anthocyanin accumulation, and derepression of light-regulated gene expression. In contrast to these morphological and gene expression changes, however, the chloroplast development program is not initiated in the dark in det2 mutants, suggesting that light-regulated gene expression precedes the differentiation of etioplasts to chloroplasts. det2 mutations thus reveal at least two classes of downstream light-regulated responses that differ in their timing and control mechanisms. Homozygous det2 mutations also affect photoperiodic responses in light-grown plants, including timing of flowering, dark adaptation of gene expression, and onset of leaf senescence. The phenotype of det1 det2 double mutants is additive, implying that DET1 and DET2 function in distinct pathways that affect downstream light-regulated genes. Furthermore, these pathways are not utilized solely during early seedling development but must also be required to regulate different aspects of the light developmental program during later stages of vegetative growth.     

Wound-Associated Competency Factors Are Required for the Proximal Cell Responses of Soybean to the Phytophthora sojae Wall Glucan Elicitor

Intact soybean (Glycine max L. [Merr.]) tissues show distinct proximal and distal cell responses to the Phytophthora sojae (Kauf. and Gerde.) wall glucan elicitor. Proximal cells respond with accumulations of glyceollin and phenolic polymers, whereas distal cells respond with an increase of isoflavone conjugates. Comparison of the activities of the P. sojae glucan in the classical cut cotyledon and a cotyledon infiltration assay suggests that the proximal, but not the distal, responses to elicitor require tissue wounding. Washing the surface of cut cotyledons prior to elicitor treatment also greatly diminishes the proximal responses, which can be restored in a dose-dependent manner by prior treatment of the washed cells with wound exudate from cut "donor" cotyledons. Thus, discrete wound-associated factors, which we term elicitation competency factors, are required for the proximal cell response to the glucan elicitor. The wound factors induce a competent state that is transient in nature. Maximal elicitor response is seen 2 to 3 h after wounding, and cells become elicitor nonresponsive after 4 h. Competency is markedly affected by the age of tissues; cotyledons become more inherently competent as they approach senescence. The time course of attainment of the competent state and its duration are strongly affected by light and temperature. Since the wound-associated competency factors can also be obtained from washings of hypersensitive lesions, we hypothesize that similar competency factors may be released from hypersensitively dying cells in incompatible infections. This event may program the immediately surrounding cells to make them competent for the proximal defense responses.     

Brassinosteroid Mutants Uncover Fine Tuning of Phytochrome Signaling

Phytochromes (phy) A and B provide higher plants the ability to perceive divergent light signals. phyB mediates red/far-red light reversible, low fluence responses (LFR). phyA mediates both very-low-fluence responses (VLFR), which saturate with single or infrequent light pulses of very low fluence, and high irradiance responses (HIR), which require sustained activation with far-red light. We investigated whether VLFR, LFR, and HIR are genetically coregulated. The Arabidopsis enhanced very-low-fluence response1 mutant, obtained in a novel screening under hourly far-red light pulses, showed enhanced VLFR of hypocotyl growth inhibition, cotyledon unfolding, blocking of greening, and anthocyanin synthesis. However, eve1 showed reduced LFR and HIR. eve1 was found allelic to the brassinosteroid biosynthesis mutant dim/dwarf1. The analysis of both the brassinosteroid mutant det2 in the Columbia background (where VLFR are repressed) and the phyA eve1 double mutant indicates that the negative effect of brassinosteroid mutations on LFR requires phyA signaling in the VLFR mode but not the expression of the VLFR. Under sunlight, hypocotyl growth of eve1 showed little difference with the wild type but failed to respond to canopy shadelight. We propose that the opposite regulation of VLFR versus LFR and HIR could be part of a context-dependent mechanism of adjustment of sensitivity to light signals.     

SPA1, a component of phytochrome A signal transduction,regulates the light signaling current

Mutations in a component of phytochrome A (phyA)-specific light signal transduction, SPA1, result in enhanced responsiveness of Arabidopsis seedlings to red and far-red light. Here, we have examined the effects of spa1 mutations on the two known modes of phyA function, the high-irradiance responses (HIRs) to continuous irradiation with far-red light and the very-low-fluence responses (VLFRs) to inductive pulses of light that establish only a small proportion of active phyA. spa1 mutants exhibited an enhanced VLFR under hourly pulses of far-red light for hypocotyl growth inhibition, cotyledon unfolding, anthocyanin accumulation, block of greening in subsequent white light and negative regulation of phyB signaling. We provide evidence that the phenotype of spa1 mutants in red light is also caused by an increase in the VLFR. Taken together, our results indicate that light-induced hypocotyl growth inhibition in spa1 mutants is primarily due to a VLFR. While wild-type seedlings required hourly pulses of far-red light to induce a VLFR, infrequent irradiation with far-red pulses (every 12 h) was sufficient to induce a strong VLFR of hypocotyl elongation in spa1 mutants. This shows that the effect of the VLFR was more persistent in spa1 mutants than in the wild type. We, therefore, propose that SPA1 has an important function in reducing the persistence of phyA signaling. spa1 mutations also enhanced the HIRs of anthocyanin accumulation and of phyA-mediated responsivity amplification towards phyB. Thus, our results suggest that spa1 mutations amplify both the phyA-mediated VLFR and the HIR.     

The VLF loci, polymorphic between ecotypes Landsberg erecta and Columbia, dissect two branches of phytochrome A signal transduction that correspond to very-low-fluence and high-irradiance responses

Phytochromes play a key role in the perception of light signals by plants. In this study, the three classical phytochrome action modes, i.e. very-low-fluence responses (VLFR), low-fluence responses (LFR) and high-irradiance responses (HIR), were genetically dissected using phyA and phyB mutants of Arabidopsis thaliana (respectively lacking phytochrome A or phytochrome B) and a polymorphism between ecotypes Landsberg erecta and Columbia. Seed germination and potentiation of greening, hypocotyl growth inhibition and cotyledon unfolding in etiolated seedlings of the ecotype Landsberg erecta showed biphasic responses to the calculated proportion of active phytochrome established by one light pulse or repeated light pulses. The first phase, i.e. the VLFR, was absent in the phyA mutant, normal in the phyB mutant (both in the Landsberg erecta background) and severely deficient in Columbia. The second phase, i.e. the LFR, was present in the phyA mutant, deficient in the phyB mutant and normal in Columbia. Under continuous far-red light, HIR of etiolated seedlings were absent in phyA and normal in phyB and Columbia. The segregation of VLFR in recombinant inbred lines derived from a cross between Landsberg erecta and Columbia was analysed by MAPMAKER/QTL. Two quantitative trait loci, one on chromosome 2 ( VLF1 ) and another on chromosome 5 ( VLF2 ), were identified as responsible for the polymorphism. Phytochrome A is proposed to initiate two transduction pathways, VLFR and HIR, involving different cells and/or different molecular steps. This is the first application of the analysis of quantitative trait loci polymorphic between ecotypes to dissect transduction chains of environmental signals.     

Photoregulation of morphogenesis of tobacco plants transformed with the gene of human interlenkin-18

The effects of blue light (BL), green light (GL), and red light (RL) on morphogenesis photoregulation of transgenic tobacco (Nicotiana tabacum L.) plants containing a copy of human interleukin-18 gene were studied. Wild-type and transgenic (Il1 No.87–1 and Il18 No.7–11) lines and derived calluses were used. Photomorphogenesis of transgenic lines under GL and RL differed substantially from that of initial line at early stages of morphogenesis; this was expressed in hypocotyl lengthening under GL and cotyledon enhanced expansion under RL. Growth responses to light quality were related to changes in the levels of zeatin, IAA, and ABA. Thus, hypocotyl lengthening and increased cotyledon area under GL occurred at the elevated level of IAA and reduced level of ABA. Growth of callus cells in transgenic lines during zero passage differed from wild-type calluses only in darkness. The data obtained indicate that tobacco plant transformation with the human interleukin-18 gene changed functioning of the photoregulation systems at early developmental stages. This phenomenon is evidently explained by the pleiotropic effects of the gene inserted.     

Light induction of cell type differentiation and cell-type-specific gene expression in cotyledons of a C(4) plant, Flaveria trinervia

In Flaveria trinervia (Asteraceae) seedlings, light-induced signals are required for differentiation of cotyledon bundle sheath cells and mesophyll cells and for cell-type-specific expression of Rubisco small subunit genes (bundle sheath cell specific) and the genes that encode pyruvate orthophosphate dikinase and phosphoenolpyruvate carboxylase (mesophyll cell specific). Both cell type differentiation and cell-type-specific gene expression were complete by d 7 in light-grown seedlings, but were arrested beyond d 4 in dark-grown seedlings. Our results contrast with those found for another C(4) dicot, Amaranthus hypochondriacus, in which light was not required for either process. The differences between the two C(4) dicot species in cotyledon cell differentiation may arise from differences in embryonic and post-embryonic cotyledon development. Our results illustrate that a common C(4) photosynthetic mechanism can be established through different developmental pathways in different species, and provide evidence for independent evolutionary origins of C(4) photosynthetic mechanisms within dicotyledonous plants.     

Functional characterization of phytochrome interacting factor 3 in phytochrome-mediated light signal transduction

Phytochromes regulate various light responses through their interactions with different signaling proteins, such as phytochrome interacting factor 3 (PIF3). However, the physiological functions of PIF3 in light signaling are not yet fully understood. To increase our understanding of these roles, we characterized a T-DNA insertional pif3 mutant and transgenic plants overexpressing the full-length PIF3. Transgenic overexpressing lines displayed longer hypocotyls and smaller cotyledons under red light and reduced cotyledon opening under both red and far-red light, whereas the pif3 mutant showed the opposite phenotypes. The accumulation of anthocyanin and chlorophyll further indicated complicated features of PIF3 function. The accumulation of anthocyanin was increased and the content of chlorophyll was decreased in the overexpression lines. Our data indicate that PIF3 plays complex roles depending on the type of light response and the light conditions.     

枣树体细胞胚发生和组织学研究

以临泽小枣子叶切块为外植体,在附加０．２mg/L IBA 1．0mg/L 6-BA的ＭＳ培养基上１周后切块边缘可诱导出白色胚愈伤组织,继续培养１个月后愈伤组织中产生体细胞胚。体细胞胚发生不同步,经历球形胚、心形胚、子叶胚等阶段,与合子胚发育途径相似。组织切片表面胚性愈伤组织细胞体积小,细胞核大、细胞质浓,细胞排列紧密;而非胚性愈伤组织细胞体积大、细胞核小、细胞质稀薄,子叶胚时期体细胞胚内部出现维管束,并观察到螺纹导管。     

Genetic identification of FIN2, a far red light-specific signaling component of Arabidopsis thaliana

Phytochrome A (PhyA) mediates most, if not all various plant responses to far-red (FR) light. Here, we report a novel genetic mutation that impairs a variety of responses in the PhyA-signaling pathway of Arabidopsis thaliana . The mutation was isolated by screening seedlings that show reduced sensitivity to continuous far-red (FRc) light irradiation, but not to continuous red (Rc) light irradiation. The mutation named fin2–1 is not allelic to a PHYA mutation. Furthermore, immunoblot analysis indicated that the amount of the phytochrome A apoprotein in the fin2–1 mutant was comparable to that in wild type. Seedling of the fin2–1 mutant showed defects in hypocotyl growth inhibition and apical hook and cotyledon opening in FRc light but not in Rc light. The results showed that the mutation occurred in a downstream signaling component potentially specific to PhyA. Other PhyAmediated responses such as FR-preconditioned blocking of greening, anthocyanin accumulation, reduction of gravitropic response, and expression of the CAB and CHS genes were impaired by the fin2–1 mutation: the degree of the mutant effect on the responses was variable. However, FR light-mediated seed germination and photoperiodic flowering responses were not affected significantly in the mutant. These results showed that FIN2 defines an upstream branch point in the PhyA signaling pathway.     

Differential DNA Endoreduplication and Protein Profile during Cotyledon Ontogeny of Vigna radiata

Differential regeneration response of the two cotyledon types. ‘Cot E’ (attached to the embryo) and ‘Cot’, of Vigna radiata have been reported earlier. The present preliminary study addresses V. radiata cotyledon development with respect to patterns of endoreduplication and protein accumulation. In this communication two distinct types of cotyledon (in relation to their attachment with the embryonal axis), differing in regeneration responses, were characterized in terms of polyploidy levels and profiles, and extent of protein synthesis/accumulation. The embryo development was studied histologically from the first day after fertilization till seed coat formation and divided into 8 different stages to determine stages of cotyledon development. Early cotyledonary stage of embryo was recorded on the 6 DAF, at this stage ‘Cot’ and ‘Cot E’ were inseparable and referred to as stage VI. ‘Cot’ and ‘Cot E’ could be distinguished from 9 DAF onwards. Two major events, endoreduplication of DNA and protein synthesis/accumulation that occur during the cotyledon development of grain-legumes, were analysed to probe the differential status, if any, in these two cotyledon types. The cell division phase of cotyledon development continues upto stage VII, while cell expansion phase starts at the stage VIII. The cotyledonary cells began to undergo the endoreduplicating cell cycle (ECC) from stage VII and continue until seed maturity. During the cell division phase the mitotic cycle and ECC occur simultaneously; whereas, only ECC continues in the cell expansion phase. Analysis of protein content indicated that ‘Cot E’ always contained comparatively higher amount during in vivo development than that of ‘Cot’. Similarity indices between ‘Cot’/’Cot E’ were 46.15%,82.35% and 90.9% at stage VII, stage VIII and at maturity, respectively, as computed from the presence oftotal polypeptides. The differential temporal pattern of DNA-endoreduplication and storage protein accumulation clearly dictates the influence of differential gene expression and regulation control in the developmental- type determination of the two cotyledons.     

Comparison of Three Phytochrome-mediated Processes in the Hypocotyl of Mustard

Anthocyanin synthesis, hair formation, and the synthesis of ascorbic acid oxidase are all phytochrome-mediated reactions occurring in the hypocotyl of mustard (Sinapis alba L.), controlled by phytochrome actually located in the hypocotyl. A comparison of these three reactions showed that in certain respects they differ greatly in their response to light. The ability of the seedling to respond to light by showing the three responses was strongly influenced by the state of development of the seedling. White light given very early after seed imbibition was unable to evoke any of the three reactions. By 50 hours after imbibition, all systems were fully inducible by light. The addition of actinomycin D to a fully competent seedling coincident with illumination strongly inhibited the development of all three responses. In contrast, the addition of cordycepin at this time inhibited the synthesis of anthocyanin and ascorbic acid oxidase but had no effect on hair formation. Cycloheximide inhibited all three responses when given up to several hours after light. This suggests the necessity for RNA and protein synthesis for light-induced expression of these reactions, and that the RNA species involved in the three reactions may have differing degrees of polyadenylation. The lag period between the onset of light and the first display of the response was 3 hours for anthocyanin and ascorbic acid oxidase synthesis, and about 5 hours for hair formation. Amounts of light sufficient to give large increases in the levels of ascorbic acid oxidase and hair formation gave a much smaller increase in anthocyanin synthesis. Hair formation and ascorbic acid oxidase synthesis showed a much greater sensitivity to induction at early stages of seedling development than did anthocyanin synthesis. Following an inductive light period, anthocyanin synthesis was sensitive to far red light inhibition for a period twice as long as the other two reactions. The differences in the response of the three reactions to light suggest that the phytochrome-mediated reactions which control their development also differ.     

Cell kinetics, stomatal differentiation, and diurnal rhythm in Allium cepa

Cell kinetics parameters were obtained for the three mitotic divisions leading to formation of stomata in the epidermis of the cotyledon of Allium cepa seedlings. Analysis of mitotic frequencies throughout the course of development showed that the asymmetrical divisions started at about 50 hr after germination, and the symmetrical divisions were first seen a few hours later. Guard mother cell divisions started around 70 hr after germination. The maximal frequency of both symmetrical and asymmetrical division was found between 3 and 5 days after germination, and the highest frequencies of GMC divisions were observed between 6 and 8.5 days. All divisions ceased after 11 days. The three cell populations analyzed displayed diurnal fluctuations of their mitotic frequencies which were characteristic of the type of cell division measured and seemed independent of the region of the cotyledon in which they took place. The symmetrical divisions displayed two diurnal peaks—one at about 0400 and the other at 1600 hr—and the asymmetrical mitoses showed a single peak at about 2200 hr. Atypical asymmetrical divisions were observed in some guard mother cells, suggesting a different developmental sequence for some of the stomatal complexes.     

Cell division and cell enlargement in isolated Cucurbita cotyledons grown in darkness and in light

The spatial and temporal patterns of post-embryonal cell growth and cell division were characterised in excised cotyledons of vegetable marrow (Cucurbita pepo L. var. giromontia Alef.) incubated in water. The concurrent roles of these two processes in cotyledon growth were determined using paradermal sections of the first palisade layer of developing cotyledons. Tissue specificity was observed in the pattern of cell division. The daughter cells derived from an initial cell, which had already differentiated before imbibition of the seeds, were tightly packed in a cluster, which enabled us to monitor cell division during early cotyledon development. Heterogeneity of cell size was recognised during the process of cell proliferation in the cluster, suggesting that cell division is uncoupled from control of cell size. There was significantly more cell division in the marginal part of the cotyledons than in other parts, suggesting high activity of the marginal meristem. Light enhanced cell and cotyledon enlargement, but had no effect on the number of divisions. This study elucidated the cellular basis of post-germinative Cucurbita cotyledon morphogenesis and development. Electronic Publication     

若干因子对鸡冠花悬浮培养中花色素苷积累的影响

本文研究几种植物生长调节剂和蔗糖浓度对鸡冠花细胞悬浮培养中花色素苷积累的影响。结果表明,细胞分裂素KT使花色素苷积累明显高于6-BA,且KT在2 μmol/L时积累量最高;2,4-D在2 μmol/L时对花色素苷积累效果明显,其它浓度的2,4-D和NAA对花色素苷积累效果不明显。高浓度蔗糖有利于花色素苷积累;MS+2,4-D(2 μmol/L)+KT(2 μmol/L)+蔗糖(292 mmol/L)为鸡冠花悬浮细胞培养生产花色素苷的最佳培养基。研究中还发现,在黑暗条件培养下无花色素苷积累,推断光是诱导花色素苷积累的主要因素。随着继代次数的增加,花色素苷含量明显增高,但到第4代时基本稳定。     

PNZIP Is a Novel Mesophyll-Specific cDNA That Is Regulated by Phytochrome and a Circadian Rhythm and Encodes a Protein with a Leucine Zipper Motif

Single, double, and triple null combinations of Arabidopsis mutants lacking the photoreceptors phytochrome (phy) A (phyA-201), phyB (phyB-5), and cryptochrome (cry) 1 (hy4-2.23n) were examined for de-etiolation responses in high-fluence red, far-red, blue, and broad-spectrum white light. Cotyledon unhooking, unfolding, and expansion, hypocotyl growth, and the accumulation of chlorophylls and anthocyanin in 5-d-old seedlings were measured under each light condition and in the dark. phyA was the major photoreceptor/effector for most far-red-light responses, although phyB and cry1 modulated anthocyanin accumulation in a phyA-dependent manner. phyB was the major photoreceptor in red light, although cry1 acted as a phyA/phyB-dependent modulator of chlorophyll accumulation under these conditions. All three photoreceptors contributed to most blue light deetiolation responses, either redundantly or additively; however, phyB acted as a modulator of cotyledon expansion dependent on the presence of cry1. As reported previously, flowering time in long days was promoted by phyA and inhibited by phyB, with each suppressing the other's effect. In addition to the effector/modulator relationships described above, measurements of hypocotyls from blue-light-grown seedlings demonstrated phytochrome activity in blue light and cry1 activity in a phyAphyB mutant background.     

Attenuation of incident light in Galax urceolata (Diapensiaceae): concerted influence of adaxial and abaxial anthocyanic layers on photoprotection

Although anthocyanin coloration in lower (abaxial) leaf cells has been documented for numerous species, the functional significance of this character has not been comprehensively investigated according to habitat or leaf orientation. Here, we demonstrate that abaxial anthocyanin may function as a photoprotectant, similarly to its purported role in upper (adaxial) cells, in leaves vulnerable to high irradiance incident on abaxial surfaces. Spectral scans were derived for Galax urceolata leaves with the following phenotypes: abaxial or adaxial anthocyanin only, abaxial and adaxial anthocyanin, and no anthocyanin. To determine whether anthocyanins conferred protection from photoinhibition, maximum photosystem II efficiencies of red (anthocyanic) and green (acyanic) surfaces were compared during and after exposure to photoinhibitory conditions. Leaves were either positioned with their adaxial surfaces facing the light source or inverted to expose abaxial surfaces. Spectral scans showed increased absorptance of 500-600 nm wavelengths by red surfaces (consistent with the absorbance spectrum of anthocyanin), regardless of whether that surface was abaxial or adaxial. Leaves with anthocyanin in either illuminated surface were also photoinhibited less than leaves lacking anthocyanin in that surface. These results suggest that anthocyanic layers reduce absorbed sunlight in the mesophyll not only for adaxial surfaces, but also for the abaxial. Adaxial/abaxial anthocyanin plasticity may therefore be adaptive in high-light environments or during light-sensitive developmental stages where leaf orientation and/or substrate albedo are variable.