Aurea Mutant Research Articles

Physiological and biochemical responses of photomorphogenic tomato mutants (cv. Micro-Tom) under water withholding

In addition to mediating photomorphogenesis, phytochromes are responsible for many abiotic stress responses, acting upon biochemical and molecular mechanisms of cell signaling. In this work, we measured the physiological and biochemical responses of phytochrome-mutant plants under water stress. In tomato (Solanum lycopersicum L.), the aurea mutant (au) is phytochrome-deficient and the high-pigment-1 mutant (hp1) has exaggerated light responses. We examined the effects of water withholding on water potential, leaf gas exchange, chlorophyll fluorescence, chloroplast pigment content and antioxidant enzyme activity in au and hp1 and their wild-type cultivar Micro-Tom (MT). Initial fluorescence and potential quantum efficiency of photosystem II (PSII) photochemistry were not affected by the treatment, but effective quantum yield of PSII, electron transport rate decreased and non-photochemical quenching increased significantly in MT. Under water withholding conditions, MT had higher malondialdehyde concentration than the mutants, but au had higher activities of catalase and ascorbate peroxidase compared to the other genotypes. The tolerance of mutants to the effects of water withholding may be explained by the higher activity of antioxidant enzymes in au and by a higher concentration of antioxidant compounds, such as carotenoids, in hp1.

Nitric Oxide, Ethylene, and Auxin Cross Talk Mediates Greening and Plastid Development in Deetiolating Tomato Seedlings.

The transition from etiolated to green seedlings involves the conversion of etioplasts into mature chloroplasts via a multifaceted, light-driven process comprising multiple, tightly coordinated signaling networks. Here, we demonstrate that light-induced greening and chloroplast differentiation in tomato (Solanum lycopersicum) seedlings are mediated by an intricate cross talk among phytochromes, nitric oxide (NO), ethylene, and auxins. Genetic and pharmacological evidence indicated that either endogenously produced or exogenously applied NO promotes seedling greening by repressing ethylene biosynthesis and inducing auxin accumulation in tomato cotyledons. Analysis performed in hormonal tomato mutants also demonstrated that NO production itself is negatively and positively regulated by ethylene and auxins, respectively. Representing a major biosynthetic source of NO in tomato cotyledons, nitrate reductase was shown to be under strict control of both phytochrome and hormonal signals. A close NO-phytochrome interaction was revealed by the almost complete recovery of the etiolated phenotype of red light-grown seedlings of the tomato phytochrome-deficient aurea mutant upon NO fumigation. In this mutant, NO supplementation induced cotyledon greening, chloroplast differentiation, and hormonal and gene expression alterations similar to those detected in light-exposed wild-type seedlings. NO negatively impacted the transcript accumulation of genes encoding phytochromes, photomorphogenesis-repressor factors, and plastid division proteins, revealing that this free radical can mimic transcriptional changes typically triggered by phytochrome-dependent light perception. Therefore, our data indicate that negative and positive regulatory feedback loops orchestrate ethylene-NO and auxin-NO interactions, respectively, during the conversion of colorless etiolated seedlings into green, photosynthetically competent young plants.

Biochemical and histological characterization of tomato mutants

Biochemical responses inherent to antioxidant systems as well morphological and anatomical properties of photomorphogenic, hormonal and developmental tomato mutants were investigated. Compared to the non-mutant Micro-Tom (MT), we observed that the malondialdehyde (MDA) content was enhanced in the diageotropica (dgt) and lutescent (l) mutants, whilst the highest levels of hydrogen peroxide (H(2)O(2)) were observed in high pigment 1 (hp1) and aurea (au) mutants. The analyses of antioxidant enzymes revealed that all mutants exhibited reduced catalase (CAT) activity when compared to MT. Guaiacol peroxidase (GPOX) was enhanced in both sitiens (sit) and notabilis (not) mutants, whereas in not mutant there was an increase in ascorbate peroxidase (APX). Based on PAGE analysis, the activities of glutathione reductase (GR) isoforms III, IV, V and VI were increased in l leaves, while the activity of superoxide dismutase (SOD) isoform III was reduced in leaves of sit, epi, Never ripe (Nr) and green flesh (gf) mutants. Microscopic analyses revealed that hp1 and au showed an increase in leaf intercellular spaces, whereas sit exhibited a decrease. The au and hp1 mutants also exhibited a decreased in the number of leaf trichomes. The characterization of these mutants is essential for their future use in plant development and ecophysiology studies, such as abiotic and biotic stresses on the oxidative metabolism.

Genetic analysis of a novel aurea mutant in wheat

A novel spontaneous chlorina mutation of wheat (Triticum aestivum L.) was found in 2000. Three types of phenotypes, i.e., green plant, intermediate yellow-green plants (or chlorina), and yellow plant (aurea), were observed in the selfed progenies of the mutant. The aurea plants usually died before heading stage, and the intermediate plants were viable but less vigorous and developed later than the green plant. The mutant wheat was analyzed genetically after self-pollination and crossing with normal green plants. Selfing the M1 plant (chlorina) results in 1:2:1 segregation ratio of aurea:chlorina:green plants. The progenies of M1 green plants were all green. The test cross between a chlorina plant and a normal plant (as male or female) gave a 1 normal green: l yellow-green segregation ratio. The result can be explained on the assumption that the aurea is homozygous for au and the chlorina heterozygous for Au. Based on these findings, this mutant is controlled by a partial dominant nucleus gene. The genotypes of aurea, chlorina, and normal plants are au/au, Au/au, and Au/Au, respectively.

The analysis of differential expression and cloning of genes related to raised secondary lateral veins mutant of Lycoris aurea

Lycoris aurea exhibits parallel venation, the main vein with many lateral veins in a longitudinal parallel arrangement. There are secondary lateral veins (SLV) between each longitudinal veins. In general, SLVs are not remarkable. In this paper, the material was one kind of Lycoris aurea mutant called Raised Secondary Lateral Veins mutant (RSLV), because many Raised Secondary Lateral Veins are in abaxial surface of its leaves. Its growing potential is weaker than that of wild type and its blades are very thin. Moreover, the stamens of RSLV degenerate completely. Two cDNA libraries were constructed from RSLV mutant and wild type (WT) leaves. From the libraries, 3,122 ESTs, which are longer than 100 bp each after vector sequence removed, were acquired by single-pass sequencing from the 5'end. Following a multistep selection, 512 70-mer oligo-DNA probes were designed for attachment on the microarray slide based on the ESTs. The gene expression profile of RSLV mutant and WT leaves was compared through the microarray at transcriptional level. The microarray experiment results were further confirmed by Quantitative Real-Time PCR (QRT-PCR). We identified 5 genes whose expressions changed more than 2-fold between RSLV mutant and WT leaves. They encode phloem protein 2 (PP2), ferritin, pectin methyl esterase (PME), chlorophyll a/b binding protein (CAB protein) and pyruvate decarboxylase (PDC), respectively. Furthermore, the full-length cDNA sequences of the 5 genes were separately obtained from RSLV and WT by RACE. The relationship between differential expressions of the genes and the formation of the RSLV mutant phenotype were discussed.

The Tomato Photomorphogenetic Mutant, aurea, is Deficient in Phytochromobilin Synthase for Phytochrome Chromophore Biosynthesis

The aurea mutants of tomato have been widely used as phytochrome-deficient mutants for photomorphogenetic and photobiological studies. By expressed sequence tag (EST)-based screening of sequence databases, we found a tomato gene that encodes a protein homologous to Arabidopsis HY2 for phytochromobilin synthase catalyzing the last step of phytochrome chromophore biosynthesis. The tomato protein expressed in Escherichia coli showed phytochromobilin synthase activity. The corresponding loci in all aurea mutants tested have nucleotide substitutions, deletions or DNA rearrangements. These results indicate that aurea is a mutant of phytochromobilin synthase in tomato. We also discuss a phylogenetic analysis of phytochromobilin synthases in the bilin reductase family.

Regeneration of protochlorophyllide in green and greening leaves of plants with varying proportions of protochlorophyllide forms in darkness

During illumination of dark‐grown plants protochlorophyllide (Pchlide) is continuously transformed to chlorophyllide (Chlide). Different dark‐grown plants, maize (Zea mays cv. Sundance), wheat (Triticum aestivum cv. Kosack), pea (Pisum sativum cv. Kelwedon wonder), the lip1 mutant of pea, and the aurea mutant of tomato (Solanum lycopersicum), have various ratios of spectral Pchlide forms in darkness. When the plants were illuminated and then returned to darkness Pchlide re‐accumulated. The proportions of different Pchlide forms within the pool of re‐accumulated Pchlide were followed by low temperature fluorescence emission and excitation spectra in green and greening leaves. After 1 h of illumination the spectral characteristics of regenerated Pchlide forms mirrored those of Pchlide in dark‐grown plants and were thus species dependent. After a prolonged illumination period (24 h) as well as in fully green leaves energy transfer to chlorophyll (Chl) masked the presence of long‐wavelength Pchlide in the fluorescence emission spectra. However, excitation spectra showed Pchlide absorption around 650 nm and its flash‐induced disappearance confirmed its nature of phototransformable Pchlide. In fact the excitation spectra showed that the proportions of different Pchlide forms in green leaves highly resembled the proportions of Pchlide forms in dark‐grown leaves and were specific for the plant variety. Thus Chl formation in both dark‐grown and light‐grown leaves can occur in a similar way through the main photoactive long‐wavelength form of Pchlide.

Involvement of phytochrome in regulation of transpiration: red-/far red-induced responses in the chlorophyll-deficient mutant of pea.

Transpiration rhythmicity and intensity were investigated in the chlorophyll-deficient mutant XL18 of Pisum sativum L. and in the phytochrome-deficient mutant aurea of Lycopersicon esculentum Mill. A custom-built psychrometer was used. In the XL18 mutant an acute transpiration response to monochromatic irradiation was observed such that red (R) light increased and far-red (FR) decreased transpiration rate, with equal rates of change. This result indicates that phytochrome is involved in regulation of transpiration. In wild type pea the chlorophyll-dependent component of transpiration was also shown to involve phytochrome. Monochromatic irradiation by red or far-red light induced an increase in transpiration with acceleration dependent on time of day. The response was irreversible by light of either wavelength. We conclude that both photoreceptors are involved in the acute response. Investigation of the daily course of transpiration revealed rhythmic changes in wild type pea and tomato under natural light conditions and in constant darkness. The rhythm was not apparent in the XL18 mutant in constant darkness, or in the aurea mutant under natural illumination. The latter results show that phytochrome, as a photoreceptor, is essential for maintaining the rhythm upon irradiation, while the photosynthetic component is crucial in darkness.

Analysis of protochlorophyllide reaccumulation in the phytochrome chromophore-deficient aurea and yg-2 mutants of tomato by in vivo fluorescence spectroscopy.

The aurea and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum) are unable to synthesize the phytochrome chromophore from heme resulting in a block of this branch of the tetrapyrrole pathway. We have previously shown that these mutants also exhibit an inhibition of protochlorophyllide (Pchlide) synthesis and it has been hypothesised that this is due to feedback inhibition by heme on the synthesis of 5-aminolevulinic acid (ALA). In this study we have investigated Pchlide reaccumulation in cotyledons from etiolated wild-type (WT), aurea and yg-2 seedlings using low-temperature fluorescence spectroscopy. WT cotyledons showed two characteristic Pchlide emission maxima at 630 nm (F630) and 655 nm (F655) respectively, while the aurea and yg-2 mutants contained only phototransformable Pchlide F655. Following a white-light flash to WT cotyledons, reaccumulation of phototransformable Pchlide F655 in the first 30 min was absolutely dependent on the presence of Pchlide F630 before the flash. Reaccumulation of Pchlide F630 was not apparent until at least 2 h after the phototransformation. In contrast, Pchlide F630 never accumulated in aurea cotyledons. The relative rates of both Pchlide F655 and total Pchlide synthesis were approximately twice as high in WT compared to aurea. Measurement of ALA synthesis capacity during this period showed that the reduced rate of Pchlide reaccumulation in aurea was due to an inhibition at this step of the pathway. In addition, feeding of ALA resulted in a substantial and equal increase of non-phototransformable Pchlide in both WT and aurea indicating that aurea cotyledons are capable of accumulating high levels of Pchlide that is not associated to the active site of NADPH:Pchlide oxidoreductase (POR). The implications of these results for the mechanism of inhibition of Pchlide synthesis in phytochrome chromophore-deficient mutants and the role of non-phototransformable Pchlide F630 during plastid development are discussed.

Evidence against the involvement of phytochrome in UVB-induced inhibition of stem growth in green tomato plants.

The effects of UVB on the kinetics of stem elongation of wild type (WT) and photomorphogenic mutants of tomato were studied by using linear voltage transducers connected to a computer. Twenty-one or twenty-six-day-old plants, grown in 12 h white light (150 mumol m(-2) s(-1) PAR)/12 h dark cycles, were first transferred to 200 mumol m(-2) s(-1) monochromatic yellow light for 12 h, then irradiated with 0.1 or 4.5 mumol m(-2) s(-1) UVB for 12 h and finally kept in darkness for another 24 h. The measurements of the kinetics of stem elongation started after 4 h under yellow light. Significant differences in stem growth during the irradiation with yellow light, as well as during the dark period, were found between the genotypes. In darkness, the magnitude of stem growth followed the order: tri > AC = fri > MMau > hp1. Two factors determined the large differences of growth in darkness: 1) the different stem elongation rate (SER) and 2) the different duration of the growing phase among the genotypes. In darkness the stem growth of au and hp1 mutants lasted for about 18 h, whereas it continued for the whole experimental period (36 h) in the other genotypes. UVB irradiation substantially reduced elongation growth of all genotypes (4.5 mumol m(-2) s(-1) being more effective than 0.1 mumol m(-2) s(-1)). Both fluence rates of UVB induced a detectable reduction of SER already after 15 min of irradiation. Red light inhibited, while far red light promoted stem growth of all the genotypes tested. fri (phyA null), tri (phyB1 null), hp1 (exhibiting exaggerated phytochrome responses) mutants and WT tomato showed similar levels of UVB-induced inhibition of growth, while the aurea mutant showed the largest growth inhibition during the 12 h of irradiation. These results indicate that phytochrome is not directly involved in UVB control of stem elongation. The results of dichromatic irradiations UVB + red or UVB + far red indicate the presence of distinct and additive action of UVB photoreceptor and of the phytochrome system in the photoregulation of stem growth.

Changes in Blue-Green and Chlorophyll Fluorescence Emission and Fluorescence Ratios during Senescence of Tobacco Plants

Blue-green fluorescence and the red/far-red chlorophyll (Chl) fluorescence emission spectra of green tobacco leaves and of aurea leaves in different stages of senescence were measured using excitation at 355 nm in order to test the suitability of the fluorescence ratios green/red and green/far-red together with other ratios as stress and pigment indicators. Young yellowish-green leaves of the aurea mutant tobacco showed a much higher blue-green fluorescence emission and lower chlorophyll fluorescence as compared to the green wild type tobacco. As a consequence, the fluorescence intensity ratios blue/green ( F440/ F520), blue/red ( F440/ F690), blue/far-red ( F440/ F740), green/red ( F520/ F690) and green/far-red ( F520/ F740), as determined from the measured fluorescence emission spectra of the adaxial and abaxial leaf sides, were quite different for both leaf types and were functions of the content of chlorophylls and carotenoids. As chlorophyll levels decreased in senescing leaves of the aurea tobacco plant, the Chl a/ b ratio remained high at 3.3 to 4.0, while the ratio of chlorophylls to carotenoids declined slightly. In contrast, the Chl fluorescence ratios ( R fd 690-values, F v / F m and F v / F 0) as physiological indicators continuously declined, indicating a successive loss of photosynthetic activity. Values of the chlorophyll fluorescence ratio F690/ F740 for adaxial and abaxial leaf-sides increased with yellowing, whereas F440/ F520 tended to decrease with the senescence-induced decline in Chl content. F440/ F690 and F520/ F690 were found to increase after an initial decrease, with leaf age and senescence. The same trend was found for F440/ F740 and F520/ F740, which exhibited maximum sensitivity with respect to Chl breakdown. Whereas the F690/ F740 ratio for abaxial versus adaxial leaf surfaces correlated linearly, with the abaxial leaf-side values always being higher than the adaxial leaf-side, values for both sides corresponded in a curvilinear fashion with leaf Chl content. Changes during early stages of leaf development were seen not only in the most commonly used red/far-red ratio, F690/ F740, but to some extent also in F440/ F520. The results indicate that changes in photosynthetic activity due to senescence and Chl breakdown can easily be detected and qualified via fluorescence ratios that are based on the UV-A excited fluorescence bands F440, F520, F690 and F740. These ratios provide a solid basis for the laser-induced remote sensing of the state of health and Chl content of vegetation. The fluorescence ratios green/red and green/far-red were found to be very suitable complementary indicators of early stress events in plants.

Photomorphogenic control of shoot regeneration from etiolated and light-grown hypocotyls of tomato

We have investigated the role of phytochrome in the photocontrol of shoot regeneration from hypocotyls of tomato ( Lycopersicon esculentum Mill.) by comparing the regenerative capability of explants obtained from etiolated and light grown seedlings of an aurea ( au) mutant and its isogenic wild type. No shoot regeneration was displayed by etiolated aurea hypocotyls. While significant regeneration was shown by etiolated wild type explants. De-etiolated explants of both genotypes were able to regenerate shoots if cultured in presence of 16 h light/8 h dark cycles. When cultured in darkness on a medium devoid of hormones no one explant regenerated shoots. Out of more than 500 aurea explants, only one differentiated a shoot under continuous red and far red light, while significant shoot regeneration from wild type hypocotyl explants was induced by both the wavebands. Hypocotyl cultures under constant red, far red and blue light showed a position-dependent capability of regenerate shoots. We propose that the phytochrome play a crucial role in shoot regeneration from hypocotyl explants of tomato. Furthermore we provide evidence that a gradient in phytochrome content between the different segments of the hypocotyl is involved in the position-dependent capability of hypocotyl cells of regenerate shoots.

Regulation of the subunit composition of plastidic glutamine synthetase of the wild-type and of the phytochrome-deficient aurea mutant of tomato by blue/UV-A- or by UV-B-light.

The photomorphogenetic aurea mutant of tomato severely deficient in spectrophotometrically active phytochromes was used to study the light-regulation of the single-copy nuclear gene encoding plastidic glutamine synthetase (GS-2; EC 6.1.3.2). The de-etiolation of dark-grown aurea mutant seedling cotyledons showed an obligatory dependency on blue light. A limited red light-responsiveness of etiolated aurea cotyledons is, however, retained as seen by the stimulation of both the GS-2 transcript and protein level in the cotyledons of aurea seedlings during growth in red light. The subunits of the octameric GS-2 enzyme were represented by polypeptides with similar electrophoretic mobilities (polypeptides a) in etiolated wild-type or aurea mutant cotyledons. GS-2 proteins with similar apparent molecular masses were also seen in the cotyledons of red light-grown aurea mutant seedlings. In contrast, GS-2 polypeptides with different apparent molecular masses (polypeptides a and b) were detected in the cotyledons of wild-type seedlings grown in red light. This difference indicates that the (post-translational) modification of tomato GS-2 subunit composition is mediated by the photoreceptor phytochrome. The illumination of etiolated wild-type or aurea cotyledons with UV-A- or UV-B-light light resulted in an increase in both the GS-2 transcript and protein level. Following illumination of etiolated wild-type seedlings with UV-A-light, the relative proportion of the GS-2 polypeptides a and b was similar than upon irradiation with blue light but different than after exposure to UV-B- or red light. This result suggests the involvement of a blue/ UV-A-light-specific photoreceptor in the regulation of tomato GS-2 subunit composition.

Kinetics of Stem Elongation in Light‐Grown Tomato Plants. Responses to Different Photosynthetically Active Radiation Levels by Wild‐Type and aurea Mutant Plants

Abstract— In this research, we measured the short‐ and long‐term, stem elongation responses of wild‐type and aurea(au) mutant tomato plants to different photosynthetically active radiation (PAR) levels by using linear voltage transducers. Stem elongation was continuously measured in green tomato plants over 2.75 days, under 12 h light/12 h dark photoperiods or in darkness after a 6 h irradiation period. There is no significant difference in stem elongation between wild‐type plants pregrown at either LOO or 400 μmol m−2 s−1 and then exposed to 12 h photoperiods. However, in the au mutant there is a very large difference between plants pregrown under 100 or 400 umol m −2 s−1 and then exposed either to 12 h photoperiods or to continuous darkness. Total stem elongation of the wild type appears to be maximal at 100 umol m−2 s−1, while that of the au mutant appears to be maximal with PAR 400 umol m−2 s−1. Wild‐type plants displayed PAR‐dependent (in the range 100‐800 umol m−2 s−1) inhibition of growth both during the day and during the night. In contrast, the au mutant showed a fluence‐rate‐dependent promotion of growth during the dark periods in the range of 10‐400 umol m−2 s−1. Large, fast and opposite changes in stem elongation rate at the light/dark and dark/light transitions were present in both genotypes. Internode elongation rate in the first half of the night was always modest in wild‐type tomato, whereas it increased rapidly in the au mutant. Stem elongation rate of wild type starts to increase after about 6 h in darkness, showing the typical time course of escape from Pfr‐mediated inhibition of elongation by an end‐of‐day response. The role of phytochrome level and type in sensing light quantity is discussed.

Simultaneous CO2- and16O2/18O2-gas exchange and fluorescence measurements indicate differences in light energy dissipation between the wild type and the phytochrome-deficientaureamutant of tomato during water stress

The CO 2 -, H 2 O- and 16 O 2 / 18 O 2 isotopic-gas exchange and the fluorescence quenching by attached leaves of the wild-type and of the phytochrome-deficient tomato aurea mutant was compared in relation to water stress and the photon fluence rate. The chlorophyll content of aurea leaves was reduced and the ultra-structure of the chloroplasts was altered. Nevertheless, the maximum rate of net CO 2 uptake in air by the yellow-green leaves of the aurea mutant was similar to that by the dark-green wild-type leaves. However, less O 2 was produced by the leaves of the aurea mutant than by leaves of the wild-type. This result indicates a reduced rate of photosynthetic electron flux in aurea mutant leaves. No difference in both photochemical and non-photochemical fluorescence quenching was found between wild-type and aurea mutant leaves. Water stress was correlated with a reversible decrease in the rates of both net CO 2 uptake and transpiration by wild-type and aurea mutant leaves. The rate of gross 16 O 2 evolution by both wild-type and aurea mutant leaves was fairly unaffected by water stress. This result shows that in both wild-type and aurea leaves, the photochemical processes are highly resistant to water stress. The rate of gross 18 O 2 uptake by wild-type leaves increased during water stress when the photon fluence rate was high. Under the same conditions, the rate of gross 18 O 2 uptake by aurea mutant leaves remained unchanged. The physiological significane of this difference with respect to the (presumed) importance of oxygen reduction in photoprotection is discussed.

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.

Phytochrome A enhances the promotion of hypocotyl growth caused by reductions in levels of phytochrome B in its far-red-light-absorbing form in light-grown Arabidopsis thaliana.

We sought to determine if phytochrome B (phyB)-mediated responses to the red light (R)/far-red light (FR) ratio are affected by phytochrome A (phyA) activity in light-grown seedlings of Arabidopsis thaliana. Pulses of FR delayed into the dark period were less effective than end-of-day (EOD) FR in promoting hypocotyl growth over a given period in darkness. White light minus blue light interposed instead of darkness between the end of the white-light photoperiod and the FR pulse was sufficient to maintain responsivity to the decrease in phyB in FR-light-absorbing form in wild-type (WT) seedlings, but not in the phyA mutant. Compared with EOD R, hourly R+FR pulses provided throughout the night caused a stronger promotion of stem growth than a single EOD R+FR pulse in WT Arabidopsis, cucumber, mustard, sunflower, tobacco, and tomato, but not in phyA Arabidopsis or in the aurea mutant of tomato. WT seedlings of Arabidopsis responded to a range of high EOD R/FR ratios, whereas the phyA mutant required stronger reductions in the EOD R/FR ratio. In sunlight, phyA seedlings of Arabidopsis showed no response to the "early warning" signals of neighboring vegetation, and hypocotyl-growth promotion occurred at higher plant densities than in the WT. Thus, under a series of light conditions, the sensitivity or responsivity to reductions in the R/FR ratio were larger in WT than in phyA seedlings. A product of phyA is therefore proposed to enhance the hypocotyl-growth response to decreases in phyB in FR-light-absorbing form in light grown seedlings.

The aurea and yellow-green-2 Mutants of Tomato Are Deficient in Phytochrome Chromophore Synthesis

The phytochrome-deficient aurea mutant of tomato has been widely used for the study of both phytochrome function and the role of other photoreceptors in the control of development in higher plants. To date the exact nature of the aurea mutation has remained unknown, though this information is clearly important for the interpretation of these studies. It has been proposed that aurea and yellow-green-2, another mutant of tomato that has a similar phenotype to aurea, could be deficient in phytochrome chromophore synthesis. We have examined this hypothesis by measuring the activity of the enzymes committed to phytochrome chromophore synthesis in these mutants. The approach takes advantage of a recently developed high pressure liquid chromatography-based assay for the synthesis of the free phytochrome chromophore, phytochromobilin from its immediate precursors biliverdin IXalpha and heme. Isolated etioplasts from aurea and yellow-green-2 seedlings were specifically unable to convert biliverdin IXalpha to 3Z-phytochromobilin and heme to biliverdin IXalpha, respectively. In addition, the level of total noncovalently bound heme in the mutants was the same as in wild type seedlings. Together, these results identify both aurea and yellow-green-2 as mutants that are deficient in phytochrome chromophore synthesis.

Analysis of phytochrome‐deficient yellow‐green‐2 and aurea mutants of tomato

Two alleles of the yellow‐green‐2 (yg‐2) and eight different alleles of the aurea (au) locus of tomato (Lycopersicon esculentum Mill.) were compared. All are characterized by a paler green colour compared with wild‐type (WT), an elongated hypocotyl in red light, and low or below detection limits of spectrophotometrically active phytochrome. Hypocotyl length was variable in white light, ranging from that of WT to more elongated. Immunochemical analysis revealed that etiolated seedlings of the yg‐2 mutant have approximately 25% of the WT level of phytochrome A protein (PHYA), whereas that of phytochrome B protein (PHYB) is normal. In this it resembles the au mutant. The au,yg‐2 double mutant has a more extreme chlorophyll deficiency than either parent. Since the yg‐2 and au mutants have a less severe phenotype at the adult stage, that is, are leaky, the additive effect can be explained by assuming that the mutants control two steps in the chromophore biosynthesis pathway. Combination, by crossing, of the yg‐2 and au mutants with a transgenic tomato line that overexpresses oat phytochrome A3 (PhyA‐3) essentially failed to restore the WT phenotype under white fluorescent light conditions, although under greenhouse conditions some evidence for increased sensitivity to light was observed. Immunochemically, oat PHYA‐3 protein is detectable in both the yg‐2,PhyA‐3 and au,PhyA‐3‘double’ mutants. Spectrophotometrical analysis, however, revealed that holophytochrome was undetectable in the yg‐2,PhyA‐3 and au,PhyA‐3‘double’ mutants. These results are compatible with both mutants being disturbed in phytochrome chromophore biosynthesis.

Non-Destructive Determination of Chlorophyll Content of Leaves of a Green and an Aurea Mutant of Tobacco by Reflectance Measurements

Summary Reflectance spectra from 400 to 800 nm with a spectral resolution of 1 nm and the content of photosynthetic pigments were acquired for leaves of an aurea mutant and a green tobacco ( Nicotiana tabacum L.) covering a range of chlorophyll a content from 7 to 30.8 μ cm -2 and total chlorophyll a + b level from 8.4 to 41.4 μg cm -2 leaf area. At a chlorophyll a content of more than 7 μg cm -2 leaf area the reflectance near 670 to 680 nm was not sensitive to a variation in chlorophyll content due to saturation of the relationship ≪absorption vs. chlorophyll content≫. The wavelength position of the red reflectance minimum (Rmin) in the main red absorption bands of in vivo chlorophyll a proved to be independent of the chlorophyll content of leaves and thus is not suitable for chlorophyll determination. Maximum sensitivity of leaf reflectance to variation in chlorophyll content was, however, found in a wide spectral range from 530 to 630 nm and near 700 nm. The wavelength of the red edge position of the reflectance spectrum (inflection point IP) in the range of 694 to 706 nm correlated very closely with the leaves' chlorophyll content in a curvilinear manner. The Ip is also closely correlated in a linear manner with the reflectance signals at 550 and 700 nm. Reflectances near 700 nm and 550 nm were found to be sensitive indicators of the red edge position and the chlorophyll content of leaves as well. The ratios of reflectances in the near infra-red range of the spectrum (above 750 nm) to that at 700 nm and at 550 nm, R NiR /R700 (or R750/R700) and R nir /R550 (or R750/R550), were directly proportional (r 2 >0.93) to the leaves' chlorophyll content. These two novel indices for chlorophyll determination (R750/R700 and R750/R550) allowed the estimation of leaf chlorophyll content with an error of less than 2.1 μg cm -2 . The two new vegetation indices, R750/R700 and R750/R550, are linearily correlated to the chlorophyll content. They exhibit a more than 6 times wider dynamic range than the widely used Normalized Difference Vegetation Index NDVT. The new vegetation index ratios are very sensitive to changes in chlorophyll content at low, medium and high chlorophyll content which is not the case for the NDVI. This suggests the application of the new indices R750/R700 and R750/R550 in the near and remote sensing of the chlorophyll content of terrestrial vegetation.