Non-yellowing Mutant Research Articles

Defect in Brnym1, a magnesium-dechelatase protein, causes a stay-green phenotype in an EMS-mutagenized Chinese cabbage (Brassica campestris L. ssp. pekinensis) line

Leaf color is an important target trait in Chinese cabbage breeding programs. Leaf yellowing may reduce crop commercial and nutritional values. Some plants with the “stay-green” trait maintain leaf greenness during senescence and even after death. Stay-green Chinese cabbage may be a focal point of future breeding projects because it could improve crop quality and yield and prolong shelf life. A new stay-green mutant, non-yellowing mutant 1 (nym1), was identified in Chinese cabbage derived from an ethyl methane sulfonate (EMS)-mutagenized population. The mutant had stay-green characteristics and a higher chlorophyll content than the wild-type during leaf senescence. The stay-green trait in the mutant Chinese cabbage was controlled by the recessive gene Brnym1. MutMap and KASP analyses showed that Brnym1 (BraA03g050600.3C) encodes an mg-dechelatase (SGR protein), which might be the causal gene of the mutation in Chinese cabbage. A nonsynonymous single nucleotide base substitution (G to A) in the third exon of Brnym1 caused an amino acid substitution from L to F in the highly conserved domain of the magnesium-dechelatase. Ectopic overexpression showed that the BrNYM1 gene of wild-type Chinese cabbage complemented the SGR-defective stay-green mutant nye1-1 of Arabidopsis. The magnesium-dechelatase activity in the nym1 mutant was significantly downregulated compared to that in the wild type. Brnym1 was relatively upregulated in the mutant during late senescence, and BrNYM1 was localized to the chloroplasts. These results indicate that Brnym1 (BraA03g050600.3C) is the causal gene of the stay-green mutation and could be of particular significance in the genetic improvement of Chinese cabbage.

Identification of a novel chloroplast protein AtNYE1 regulating chlorophyll degradation during leaf senescence in Arabidopsis.

A dramatic increase of chlorophyll (Chl) degradation occurs during senescence of vegetative plant organs and fruit ripening. Although the biochemical pathway of Chl degradation has long been proposed, little is known about its regulatory mechanism. Identification of Chl degradation-disturbed mutants and subsequently isolation of responsible genes would greatly facilitate the elucidation of the regulation of Chl degradation. Here, we describe a nonyellowing mutant of Arabidopsis (Arabidopsis thaliana), nye1-1, in which 50% Chl was retained, compared to less than 10% in the wild type (Columbia-0), at the end of a 6-d dark incubation. Nevertheless, neither photosynthesis- nor senescence-associated process was significantly affected in nye1-1. Characteristically, a significant reduction in pheophorbide a oxygenase activity was detected in nye1-1. However, no detectable accumulation of either chlorophyllide a or pheophorbide a was observed. Reciprocal crossings revealed that the mutant phenotype was caused by a monogenic semidominant nuclear mutation. We have identified AtNYE1 by positional cloning. Dozens of its putative orthologs, predominantly appearing in higher plant species, were identified, some of which have been associated with Chl degradation in a few crop species. Quantitative polymerase chain reaction analysis showed that AtNYE1 was drastically induced by senescence signals. Constitutive overexpression of AtNYE1 could result in either pale-yellow true leaves or even albino seedlings. These results collectively indicate that NYE1 plays an important regulatory role in Chl degradation during senescence by modulating pheophorbide a oxygenase activity.

Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L.

The activities of chlorophyllase, contents of pigments including chlorophyll a and b, chlorophyllide a and b, and phaeophorbide a during leaf senescence under low oxygen (0.5% O 2 ) and control (air) were investigated in a non-yellowing mutant and wild-type leaves of snap beans (Phaseolus vulgaris L.). Chlorophyllase from leaf tissues had maximum activity when incubated at 40°C in a mixture containing 50% acetone. In both mutant and wild type, chlorophyllase activity was the highest in freshly harvested non-senescent leaves and decreased sharply in the course of senescence, indicating that the loss of chlorophylls in senescing leaves is not directly related to the activity of chlorophyllase and that chlorophyllase activity is not altered in the mutant. The wild type had higher ratios of chlorophyll a to chlorophyll b than the mutant and chlorophyll a : b ratios increased during senescence in both types. In the senescent mutant leaves, accumulations of chlorophyllide a and chlorophyllide b were detected, but no phaeophorbide a was found. Chlorophyllide b had a greater accumulation than chlorophyllide a in the early stage of senescence. Low oxygen treatment not only delayed chlorophyll degradation but also enhanced the accumulations of chlorophyllide a and b and lowered the ratios of chlorophyll a to chlorophyll b.

Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L

Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L

Chlorophyll breakdown in senescent leaves identification of the biochemical lesion in a stay-green genotype of Festuca pratensis Huds.

Chlorophyll breakdown in senescent leaves proceeds in essentially three steps: dephytylation by the action of chlorophyllase; conversion of chlorophyllide to phaeophorbide by Mg-dechelatase; and oxygenolytic cleavage of the chlorine-macrocycle by a newly discovered dioxygenase. The metabolic lesion responsible for high retention of chlorophyll during foliar senescence in a mutant genotype of meadow fescue (Festuca pratensis Huds.) was located in the third step of the breakdown pathway. Senescent leaves of both the normally yellowing reference genotype, c.v Rossa, and the non-yellowing mutant Bf993 were shown to be competent with regard to chlorophyllase and Mg-dechelatase. On the other hand, thylakoids isolated from senescent leaves of cv. Rossa were able to carry out oxygenolysis of phaeophorbide into a colourless fluorescent catabolite in vitro, whereas Bf993 thylakoids were deficient in this activity. It is concluded that the Sid locus, a mutant allele of which is responsible for the stay-green character, encodes or regulates the gene for, phaeophorbide a dioxygenase.

Leaf Senescence in a Non-Yellowing Mutant of Festuca pratensis. Transcripts and Translation Products

Summary Unlike leaves of the normal genotype cv. Rossa, green tissue of Bf993, a nuclear gene mutant of Festuca pratensis, does not turn yellow either during the process of sequential senescence in the intact plant or during senescence induced by detachment. Proteins of detached leaf tissue were labelled with [35S]-methionine and analysed by electrophoresis and fluorography. Many novel proteins were made during senescence and there were several clear genotypic differences. The nature and abundances of gene transcripts in senescing detached and attached leaves were analysed by translation in a cell free system and by northern blotting. There were few discernible changes in the majority of translatable mRNAs during development and senescence of leaves on the intact plant, but a small number of new mRNA species consistently appeared de novo as a result of ageing, a few became undetectable and several others increased or decreased significantly. Northern analysis confirmed that, amongst the messages decreasing in abundance with leaf age and in isolated tissue, there are transcripts of nuclear- and plastome-encoded plastid proteins. Moreover, the previously-reported abnormal stability of intrinsic thylakoid membrane proteins in the mutant is not a consequence of abnormal synthesis or turnover of the corresponding transcripts. A consistent difference in gene expression was observed between the normal genotype and lines homozygous or heterozygous for the mutant gene: one or two characteristic polypeptides (M, 60-65 x 103) were resolved by both one- and two-dimensional electrophoresis amongst the translation products of mutant as compared with normal mRNAs. These polypeptides were also observed amongst the translation products of RNA isolated from polyribosomes, suggesting that the corresponding messages associate with, and are probably therefore translatable by, the protein synthesis machinery in vivo.

Immunochemical quantification of cytochrome f in leaves of a non-yellowing senescence mutant of Festuca pratensis

A non-competitive enzyme-linked immunosorbent assay (ELISA) which enables detection of as little as 0.1 ng cytochrome f in leaf extracts has been developed. No evidence for specific or non-specific interference by proteins other than cytochrome f was found. The assay was applied to a comparative study of age-related changes in the cytochrome f content of leaves of Festuca pratensis Huds. cv. Rossa, and a non-yellowing mutant genotype (Bf993) having a lesion in the mechanism responsible for thylakoid membrane disassembly. Cytochrome f in senescent leaves of the latter genotype was found to be present at significantly higher levels than in the wild-type, implying an inability on the part of the mutant to degrade this protein. The results obtained by ELISA were confirmed by antibody probing of Western blots.

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Proteins of photosystem II

The senescence of leaves is characterized by yellowing as chlorophyll pigments are degraded. Proteins of the chloroplasts also decline during this phase of development. There exists a non-yellowing mutant genotype of Festuca pratensis Huds. which does not suffer a loss of chlorophyll during senescence. The fate of chloroplast membrane proteins was studied in mutant and wild-type plants by immune blotting and immuno-electron microscopy. Intrinsic proteins of photosystem II, exemplified by the light-harvesting chlorophyll a/b-binding protein (LHCP-2) and D1, were shown to be unusually stable in the mutant during senescence, whereas the extrinsic 33-kilodalton protein of the oxygen-evolving complex was equally lable in both genotypes. An ultrastructural study revealed that while the intrinsic proteins remained in the internal membranes of the chloroplasts, they ceased to display the heterogenous lateral distribution within the lamellae which was characteristic of nonsenescent chloroplasts. These observations are discussed in the light of possible mechanisms of protein turnover in chloroplasts.

Photobleaching of chloroplast pigments in leaves of a non-yellowing mutant genotype of Festuca pratensis

Chloroplast pigment contents in a normal (cv Rossa and a non-yellowing mutant (Bf993 genotype of Festuca pratensis were compared during senescence of excised leaf tissue in darkness and under photobleaching conditions. Methylviologen-treated leaves of both genotypes were bleached to the same extent when illuminated. Feeding δ-aminolaevulinic acid in the dark for 24 hr followed by exposure to light also resulted in photodestruction of chlorophylls and carotenoids in both Rossa and Bf993. Mutant leaf tissue retained more than 92% of chlorophyll during eight days in continuous darkness, but the pigment became progressively more susceptible to photobleaching as senescence proceeded. The t 1/2 for loss of capacity to withstand photobleaching was 2.00 days, significantly less than that for chlorophyll degradation in senescing Rossa tissue (2.94–3.14 days). The ratios of green to yellow pigments decreased greatly during senescence of the normal genotype, but remained the same or increased during all photobleaching treatments. It is concluded that pigment degradation during senescence normally proceeds by a pathway quite separate from non-enzymic, free-radical-mediated routes exemplified by the photobleaching reaction.

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Photosynthesis and photosynthetic electron transport

The photosynthetic capacity of detached leaves of a non-yellowing mutant of Festuca pratensis Huds. declined during senescence at a similar rate to that in a normal cultivar. Respiratory oxygen uptake in the dark continued at similar rates in both genotypes during several days of senescence. In chloroplasts isolated from leaves at intervals after excision, the rate of photosystem I (PS I)-mediated methyl viologen reduction using reduced N,N,N',N'-tetramethyl-p-phenylene diamine as electron donor also declined in both genotypes, possibly due to loss of integrity of the photosynthetic apparatus in the cytochrome f-plastocyanin region. There was a similar fall in PS II electron transport using water as electron donor and measured at the rate of reduction of 2,6-dichlorophenolindophenol. Partial restoration of this activity by the addition of diphenyl carbazide was evidence for lability of the oxygen-evolving complex during senescence. An accentuated difference between mutant and normal material in this case indicated that the mutant retains a greater number of functional PS II centres. Changes in the light-saturation characteristics of the two photosystems have been discussed in relation to the organization of the photosynthetic membranes during senescence.

Immunological quantification of the chlorophyll [formula omitted] binding protein in senescing leaves of Festuca pratensis Huds.

A solid-phase immunoassay technique was used to measure the concentrations of the light-harvesting chlorophyll a b protein (LHCP) in senescing leaves of normal and non-yellowing mutant genotypes of Festuca pratensis Huds. The rapid loss of this protein occurring in the wildtype was not observed in the mutant, which retained its complement of LHCP throughout. This result is discussed in relation to the possible nature of the mutation and the normal mechanisms of thylakoid membrane disassembly during senescence.

Leaf senescence in a non-yellowing mutant of Festuca pratensis Huds.

A study was made of linolenic acid-dependent oxidative chlorophyll bleaching (CHLOX) by thylakoid membranes from senescing leaf tissue of a normal cultivar (cv. Rossa) and a non-yellowing mutant genotype (Bf 993) of Festuca pratensis Huds. To overcome the problem of variation in levels of endogenous chlorophyll substrate in membranes from different sources, light-harvesting complex (LHC) was used to supplement thylakoid pigment. It was shown that CHLOX is associated with both Photosystem I and LHC-rich thylakoid subfractions but that purified LHC has negligible associated CHLOX activity and stimulates the rate of bleaching by isolated entire chloroplast membranes. Non-senescent tissue of Bf 993 and Rossa had essentially identical thylakoid CHLOX levels, which subsequently declined during senescence in darkness. The half-life of CHLOX from the mutant was three times greater than that of the normal genotype. In both cultivars, the amount of CHLOX assayed in thylakoids isolated at different times during senescence was more than adequate to support the corresponding in-vivo rate of pigment degradation as calculated from the half-life for chlorophyll. It was concluded that the non-yellowing mutation is not expressed through a lack of CHLOX activity. The role of linolenic acid metabolism in the regulation of thylakoid structure and function during senescence, and as a likely site of the non-yellowing lesion, are discussed.

Leaf senescence in a non-yellowing mutant of Festuca pratensis

The lipid compositions of leaves from Festuca pratensis cv. Rossa (yellowing) were compared with those from a non-yellowing mutant, Bf 993. The leaves of Bf 993 contained a higher level of acyl lipids on both a fresh-weight and a dry-weight basis. Diacylgalactosylglycerol, diacylgalabiosylglycerol and phosphatidylinositol were relatively enriched in the Bf 993 mutant while phosphatidylcholine was relatively reduced. There were no differences in the fatty-acid compositions of individual lipids between the two varieties. During senescence, the lipids of cv. Rossa were progressively degraded over an 8-d period. In contrast little lipid degradation was observed in the Bf 993 mutant during the first 4 d. The results support the hypothesis that the slower senescence changes of the Bf 993 mutant may be due, in part, to an altered membrane lipid composition.

Leaf senescence in a non-yellowing mutant of Festuca pratensis

The acid proteinases (E.C. 3.4.4.?) of mature, nonsenescent Festuca pratensis leaf tissue were fractionated into one major and three minor activities by ion exchange chromatography. During senescence of leaf sections in darkness there was a general increase in total proteinase activity but no qualitative alteration in the proteinase complement. The pattern of proteinases in the nonyellowing mutant Bf 993 was essentially identical to that of the normal genotype Rossa. The pH responses of proteinases from senescent Bf 993 and Rossa tissue were measured using as substrates (14)C-labelled protein fractions prepared from barley seedlings. These fractions were: ribulose bisphosphate carboxylase; low molecular weight soluble protein; ethylene diamine tetraacetate soluble thylakoid protein; chloroform: methanol soluble thylakoid protein; and residual protein. For each substrate, the pH optimum was 5-6, there was a shoulder on the neutral-alkaline side of the pH curve and hydrolase activity was stimulated by borate at high pH. The responses of Bf 993 proteinases were very similar to those of Rossa. These results suggest that it is the accessibility of thylakoid proteins to proteinase action rather than proteinase per se that is impaired in the non-yellowing mutant.

Leaf senescence in a non-yellowing mutant of Festuca pratensis

Soluble and thylakoid membrane polypeptides from senescing leaf tissue of Rossa, a normal yellowing Festuca pratensis genotype, were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and compared with those of the non-yellowing mutant Bf 993. Subunits of ribulose-1,5-bisphosphate carboxylase were the major soluble polypeptides and declined to low levels in senescing leaves of both genotypes. The major thylakoid polypeptides were those associated with the chlorophyllprotein complexes CPI and CPII. The levels of all thylakoid polypeptide species fell during senescence of Rossa leaf tissue but Bf993 lamellae retained CPI, CPII and a number of other hydrophobic low molecular weight polypeptides. The increasing hydrophobicity and decreasing protein complement of Bf 993 thylakoids were reflected in a fall in membrane density from 1.16 to 1.13 g cm(-3) over 8 d of senescence and a decline in the extractability of chlorophyll-containing membranes in the same period. In Bf993 the molar ratio of chlorophyll to hydrophobic membrane protein increased from 92 at day 0 to 296 at day 8. In the same time the ratio for Rossa increased from 88 to 722 and 8 d-senesced Rossa tissue yielded less than 2% of the solvent-soluble protein it contained at day 0 as compared with 24% for the protein of Bf993. These results are discussed in relation to the nature of the non-yellowing lesion.

Nucleic acids from leaves of a yellowing and a non-yellowing variety of Festuca pratensis Huds.

High molecular weight ribosomal RNA components and their pattern of loss on ageing of excised leaf sections were the same in the non-yellowing mutant and the normal genotype of Festuca pratensis even though the mutant showed retarded chlorophyll loss. Thus it appears that the genetic lesion does not extend to changes in the ribosomal RNA components of chloroplasts or cytoplasm.

Ultrastructure, polypeptide composition and photochemical activity of chloroplasts during foliar senescence of a non-yellowing mutant genotype of Festuca pratensis Huds.

A study was made of the structure and function of senescent chloroplasts from a non-yellowing (NY) mutant of Festuca pratensis. Electron microscopy suggested that the stroma matrix was destroyed but that thylakoid membranes persisted in a loose, unstacked condition. By contrast, chloroplasts from the normal (Y) genotype lost both stroma and recognizable thylakoid systems. Fraction 1, the major protein of the stroma, disappeared from Y and NY at similar rates during senescence. The activities of photosystems I and II from NY also declined at a similar rate to Y photosystems. Polypeptides of chloroplast membranes were separated by SDS gel electrophoresis into at least 30 components. There was considerable heterogeneity in rates of breakdown of the different protein species of the membranes. Of the five major polypeptide components, two had kinetics of breakdown similar to those of stroma proteins and were lost from NY and Y at about the same rate, whereas the remaining three (one of which was tentatively identified as the apoprotein of the light-harvesting chlorophyll-protein complex) were more stable in NY than in Y. These results are discussed in relation to the mechanism and function of chloroplast disintegration during leaf senescence.