Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll b and light-harvesting complex II degradation during senescence in rice

Yellowing, which is related to the degradation of chlorophyll and chlorophyll–protein complexes, is a notable phenomenon during leaf senescence. NON-YELLOW COLORING1 ( NYC1 ) in rice encodes a membrane-localized short-chain dehydrogenase/reductase (SDR) that is thought to represent a chlorophyll  b reductase necessary for catalyzing the first step of chlorophyll  b degradation. Analysis of the nyc1 mutant, which shows the stay-green phenotype, revealed that chlorophyll  b degradation is required for the degradation of light-harvesting complex II and thylakoid grana in leaf senescence. Phylogenetic analysis further revealed the existence of NYC1-LIKE (NOL) as the most closely related protein to NYC1. In the present paper, the nol mutant in rice was also found to show a stay-green phenotype very similar to that of the nyc1 mutant, i.e. the degradation of chlorophyll  b was severely inhibited and light-harvesting complex II was selectively retained during senescence, resulting in the retention of thylakoid grana even at a late stage of senescence. The nyc1 nol double mutant did not show prominent enhancement of inhibition of chlorophyll degradation. NOL was localized on the stromal side of the thylakoid membrane despite the lack of a transmembrane domain. Immunoprecipitation analysis revealed that NOL and NYC1 interact physically in vitro . These observations suggest that NOL and NYC1 are co-localized in the thylakoid membrane and act in the form of a complex as a chlorophyll  b reductase in rice.     

Defect in non-yellow coloring 3, an α/β hydrolase-fold family protein, causes a stay-green phenotype during leaf senescence in rice

Chlorophyll degradation is an important phenomenon in the senescence process. It is necessary for the degradation of certain chlorophyll–protein complexes and thylakoid membranes during leaf senescence. Mutants retaining greenness during leaf senescence are known as 'stay-green' mutants. Non-functional type stay-green mutants, which possess defects in chlorophyll degradation, retain greenness but not leaf functionality during senescence. Here, we report a new stay-green mutant in rice, nyc3 . nyc3 retained a higher chlorophyll a and chlorophyll b content than the wild-type but showed a decrease in other senescence parameters during dark incubation, suggesting that it is a non-functional stay-green mutant. In addition, a small amount of pheophytin a , a chlorophyll a -derivative without Mg²⁺ ions in its tetrapyrrole ring, accumulated in the senescent leaves of nyc3 . nyc3 shows a similar but weaker phenotype to stay green ( sgr ), another non-functional stay-green mutant in rice. The chlorophyll content of nyc3 sgr double mutants at the late stage of leaf senescence was also similar to that of sgr . Linkage analysis revealed that NYC3 is located near the centromere region of chromosome 6. Map-based cloning of genes near the centromere is very difficult because of the low recombination rate; however, we overcame this problem by using ionizing radiation-induced mutant alleles harboring deletions of hundreds of kilobases. Thus, it was revealed that NYC3 encodes a plastid-localizing α/β hydrolase-fold family protein with an esterase/lipase motif. The possible function of NYC3 in the regulation of chlorophyll degradation is discussed.     

Characterization of green seed,an enhancer of abi3-1 in Arabidopsis that affects seed longevity

Seeds are usually stored in physiological conditions in which they gradually lose their viability and vigor depending on storage conditions, storage time, and genotype. Very little is known about the underlying genetics of seed storability and seed deterioration. We analyzed a mutant in Arabidopsis disturbed in seed storability. This mutant was isolated as a grs (green-seeded) mutant in an abi3-1 (abscisic acid 3) mutant background. Genetic and physiological characterization showed that the monogenic grs mutant was not visibly green seeded and mapped on chromosome 4. This enhancer mutation did not affect the ABA sensitivity of seed germination or seed dormancy but was found to affect seed storability and seedling vigor. Seed storability was assessed in a controlled deterioration test, in which the germination capacity of the mutant decreased with the duration of the treatment. The decrease in viability and vigor was confirmed by storing the seeds in two relative humidities (RHs) for a prolonged period. At 60% RH, the mutant lost germinability, but storage at 32% RH showed no decrease of germination although seed vigor decreased. The decrease in viability and vigor could be related to an increase in conductivity, suggesting membrane deterioration. This was not affected by light conditions during imbibition, expected to influence the generation of active oxygen species. During seed maturation, ABI3 regulates several processes: acquiring dormancy and long-term storability and loss of chlorophyll. Our results indicate that GRS is a common regulator in the latter two but not of dormancy/germination.     

Maternal Effects Govern Variable Dominance of Two Abscisic Acid Response Mutations in Arabidopsis thaliana

Three abscisic acid (ABA)-controlled responses (seed dormancy, inhibition of germination by applied ABA, and stomatal closure) were compared in wild-type versus homo- and heterozygotes of two Arabidopsis thaliana ABA-insensitive mutants, abi1 and abi2. We found that sensitivity of seeds to applied ABA is partially maternally controlled but that seed dormancy is determined by the embryonic genotype. The effects of the abi1 and abi2 mutations on ABA sensitivity of seed germination ranged from recessive to nearly fully dominant, depending on the parental source of the mutant allele. This maternal effect disappeared during vegetative growth. Stomatal regulation in heterozygotes showed substantial variability, but the average water loss was intermediate between that of homozygous mutants and wild type.     

NYC4, the rice ortholog of Arabidopsis THF1, is involved in the degradation of chlorophyll – protein complexes during leaf senescence

Yellowing/chlorophyll breakdown is a prominent phenomenon in leaf senescence, and is associated with the degradation of chlorophyll – protein complexes. From a rice mutant population generated by ionizing radiation, we isolated nyc4‐1, a stay‐green mutant with a defect in chlorophyll breakdown during leaf senescence. Using gene mapping, nyc4‐1 was found to be linked to two chromosomal regions. We extracted Os07g0558500 as a candidate for NYC4 via gene expression microarray analysis, and concluded from further evidence that disruption of the gene by a translocation‐related event causes the nyc4 phenotype. Os07g0558500 is thought to be the ortholog of THF1 in Arabidopsis thaliana. The thf1 mutant leaves show variegation in a light intensity‐dependent manner. Surprisingly, the F_v/F_m value remained high in nyc4‐1 during the dark incubation, suggesting that photosystem II retained its function. Western blot analysis revealed that, in nyc4‐1, the PSII core subunits D1 and D2 were significantly retained during leaf senescence in comparison with wild‐type and other non‐functional stay‐green mutants, including sgr‐2, a mutant of the key regulator of chlorophyll degradation SGR. The role of NYC4 in degradation of chlorophyll and chlorophyll – protein complexes during leaf senescence is discussed.     

Rice NON-YELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence

Chlorophyll degradation is an aspect of leaf senescence, which is an active process to salvage nutrients from old tissues. non-yellow coloring1 (nyc1) is a rice (Oryza sativa) stay-green mutant in which chlorophyll degradation during senescence is impaired. Pigment analysis revealed that degradation of not only chlorophylls but also light-harvesting complex II (LHCII)-bound carotenoids was repressed in nyc1, in which most LHCII isoforms were selectively retained during senescence. Ultrastructural analysis of nyc1 chloroplasts revealed that large and thick grana were present even in the late stage of senescence, suggesting that degradation of LHCII is required for the proper degeneration of thylakoid membranes. Map-based cloning of NYC1 revealed that it encodes a chloroplast-localized short-chain dehydrogenase/reductase (SDR) with three transmembrane domains. The predicted structure of the NYC1 protein and the phenotype of the nyc1 mutant suggest the possibility that NYC1 is a chlorophyll b reductase. Although we were unable to detect the chlorophyll b reductase activity of NYC1, NOL (for NYC1-like), a protein closely related to NYC1 in rice, showed chlorophyll b reductase activity in vitro. We suggest that NYC1 and NOL encode chlorophyll b reductases with divergent functions. Our data collectively suggest that the identified SDR protein NYC1 plays essential roles in the regulation of LHCII and thylakoid membrane degradation during senescence.     

Acquisition of Desiccation Tolerance and Longevity in Seeds of Arabidopsis thaliana (A Comparative Study Using Abscisic Acid-Insensitive abi3 Mutants)

Two new abscisic acid (ABA)-insensitive mutants of Arabidopsis thaliana affected in the abi3 locus are described. These new mutants are severely ABA insensitive. Like the earlier described abi3-1 and the ABA-deficient and -insensitive double mutant aba,abi3, these new mutants vary in the extent of ABA-correlated physiological responses. Mutant seeds fail to degrade chlorophyll during maturation and show no dormancy, and desiccation tolerance and longevity are poorly developed. Carbohydrate accumulation as well as synthesis of LEA or RAB proteins are often suggested to be essential for acquisition of desiccation tolerance. In this work two points are demonstrated. (a) Accumulation of carbohydrates as such does not correlate with acquisition of desiccation tolerance or longevity. It is suggested that a low ratio of mono- to oligosac-charides rather than the absolute amount of carbohydrates controls seed longevity or stability to desiccation tolerance. (b) Synthesis of a few assorted proteins, which is responsive to ABA in the later part of seed maturation, is not correlated with desiccation tolerance or longevity.     

Seed after-ripening is a discrete developmental pathway associated with specific gene networks in Arabidopsis

After-ripening (AR) is a time and environment regulated process occurring in the dry seed, which determines the germination potential of seeds. Both metabolism and perception of the phytohormone abscisic acid (ABA) are important in the initiation and maintenance of dormancy. However, molecular mechanisms that regulate the capacity for dormancy or germination through AR are unknown. To understand the relationship between ABA and AR, we analysed genome expression in Arabidopsis thaliana mutants defective in seed ABA synthesis (aba1-1) or perception (abi1-1). Even though imbibed mutant seeds showed no dormancy, they exhibited changes in global gene expression resulting from dry AR that were comparable with changes occurring in wild-type (WT) seeds. Core gene sets were identified that were positively or negatively regulated by dry seed storage. Each set included a gene encoding repression or activation of ABA function (LPP2 and ABA1, respectively), thereby suggesting a mechanism through which dry AR may modulate subsequent germination potential in WT seeds. Application of exogenous ABA to after-ripened WT seeds did not reimpose characteristics of freshly harvested seeds on imbibed seed gene expression patterns. It was shown that secondary dormancy states reinstate AR status-specific gene expression patterns. A model is presented that separates the action of ABA in seed dormancy from AR and dry storage regulated gene expression. These results have major implications for the study of genetic mechanisms altered in seeds as a result of crop domestication into agriculture, and for seed behaviour during dormancy cycling in natural ecosystems.     

Leafy Cotyledon Mutants of Arabidopsis

We have previously described a homeotic leafy cotyledon (lec) mutant of Arabidopsis that exhibits striking defects in embryonic maturation and produces viviparous embryos with cotyledons that are partially transformed into leaves. In this study, we present further details on the developmental anatomy of mutant embryos, characterize their response to abscisic acid (ABA) in culture, describe other mutants with related phenotypes, and summarize studies with double mutants. Our results indicate that immature embryos precociously enter a germination pathway after the torpedo stage of development and then acquire characteristics normally restricted to vegetative parts of the plant. In contrast to other viviparous mutants of maize (vp1) and Arabidopsis (abi3) that produce ABA-insensitive embryos, immature lec embryos are sensitive to ABA in culture. ABA is therefore necessary but not sufficient for embryonic maturation in Arabidopsis. Three other mutants that produce trichomes on cotyledons following precocious germination in culture are described. One mutant is allelic to lec1, another is a fusca mutant (fus3), and the third defines a new locus (lec2). Mutant embryos differ in morphology, desiccation tolerance, pattern of anthocyanin accumulation, presence of storage materials, size and frequency of trichomes on cotyledons, and timing of precocious germination in culture. The leafy cotyledon phenotype has therefore allowed the identification of an important network of regulatory genes with overlapping functions during embryonic maturation in Arabidopsis.     

In Vivo Inhibition of Seed Development and Reserve Protein Accumulation in Recombinants of Abscisic Acid Biosynthesis and Responsiveness Mutants in Arabidopsis thaliana

In Arabidopsis thaliana, seed development in recombinants of the ABA-deficient aba mutant with the ABA response mutants abi1 or abi3 is compared to wild type and the monogenic parents. Aberrant seed development occurred in the aba,abi3 recombinant and was normal in aba,abi1, abi3 and aba,abi1 seeds. Embryos of the recombinant aba,abi3 seeds maintained the green color until maturity, the seeds kept a high water content, did not form the late abundant 2S and 12S storage proteins, were desiccation intolerant, and often showed viviparous germination. Application of ABA, and particularly of an ABA analog, to the roots of plants during seed development partially alleviated the aberrant phenotype. Seeds of aba,abi3 were normal when they developed on a mother plant heterozygous for Aba. In contrast to seed development, the induction of dormancy was blocked in all monogenic mutants and recombinants. Dormancy was only induced by embryonic ABA; it could not be increased by maternal ABA or ABA applied to the mother plant. It is concluded that endogenous ABA has at least two different effects in developing seeds. The nature of these responses and of the ABA response system is discussed.     

The Arabidopsis ABA-deficient mutant aba4 demonstrates that the major route for stress-induced ABA accumulation is via neoxanthin isomers

A novel abscisic acid (ABA)-deficient mutant, aba4, was identified in a screen for paclobutrazol-resistant germination. Compared with wild-type, the mutant showed reduced endogenous ABA levels in both dehydrated rosettes and seeds. Carotenoid composition analysis demonstrated that the defective locus affects neoxanthin synthesis. The ABA4 gene was identified by map-based cloning, and found to be a unique gene in the Arabidopsis genome. The predicted protein has four putative helical transmembrane domains and shows significant similarity to predicted proteins from tomato, rice and cyanobacteria. Constitutive expression of the ABA4 gene in Arabidopsis transgenic plants led to increased accumulation of trans-neoxanthin, indicating that the ABA4 protein has a direct role in neoxanthin synthesis. aba4 mutant phenotypes were mild compared with previously identified ABA-deficient mutants that exhibit vegetative tissue phenotypes. Indeed, ABA levels in seeds of aba4 mutants were higher than those of aba1 mutants. As aba1 mutants are also affected in a unique gene, this suggests that ABA can be produced in the aba4 mutant by an alternative pathway using violaxanthin as a substrate. It appears, therefore, that in Arabidopsis both violaxanthin and neoxanthin are in vivo substrates for 9-cis-epoxycarotenoid dioxygenases. Furthermore, significantly reduced levels of ABA were synthesized in the aba4 mutant on dehydration, demonstrating that ABA biosynthesis in response to stress must occur mainly via neoxanthin isomer precursors.     

The Time Required for Dormancy Release in Arabidopsis Is Determined by DELAY OF GERMINATION1 Protein Levels in Freshly Harvested Seeds

Seed dormancy controls the start of a plant's life cycle by preventing germination of a viable seed in an unfavorable season. Freshly harvested seeds usually show a high level of dormancy, which is gradually released during dry storage (after-ripening). Abscisic acid (ABA) has been identified as an essential factor for the induction of dormancy, whereas gibberellins (GAs) are required for germination. The molecular mechanisms controlling seed dormancy are not well understood. DELAY OF GERMINATION1 (DOG1) was recently identified as a major regulator of dormancy in Arabidopsis thaliana. Here, we show that the DOG1 protein accumulates during seed maturation and remains stable throughout seed storage and imbibition. The levels of DOG1 protein in freshly harvested seeds highly correlate with dormancy. The DOG1 protein becomes modified during after-ripening, and its levels in stored seeds do not correlate with germination potential. Although ABA levels in dog1 mutants are reduced and GA levels enhanced, we show that DOG1 does not regulate dormancy primarily via changes in hormone levels. We propose that DOG1 protein abundance in freshly harvested seeds acts as a timer for seed dormancy release, which functions largely independent from ABA.     

Gibberellin requirement for Arabidopsis seed germination is determined both by testa characteristics and embryonic abscisic acid

The mechanisms imposing a gibberellin (GA) requirement to promote the germination of dormant and non-dormant Arabidopsis seeds were analyzed using the GA-deficient mutant ga1, several seed coat pigmentation and structure mutants, and the abscisic acid (ABA)-deficient mutant aba1. Testa mutants, which exhibit reduced seed dormancy, were not resistant to GA biosynthesis inhibitors such as tetcyclacis and paclobutrazol, contrarily to what was found before for other non-dormant mutants in Arabidopsis. However, testa mutants were more sensitive to exogenous GAs than the wild-types in the presence of the inhibitors or when transferred to a GA-deficient background. The germination capacity of the ga1-1 mutant could be integrally restored, without the help of exogenous GAs, by removing the envelopes or by transferring the mutation to a tt background (tt4 and ttg1). The double mutants still required light and chilling for dormancy breaking, which may indicate that both agents can have an effect independently of GA biosynthesis. The ABA biosynthesis inhibitor norflurazon was partially efficient in releasing the dormancy of wild-type and mutant seeds. These results suggest that GAs are required to overcome the germination constraints imposed both by the seed coat and ABA-related embryo dormancy.     

Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis

The testa of higher plant seeds protects the embryo against adverse environmental conditions. Its role is assumed mainly by controlling germination through dormancy imposition and by limiting the detrimental activity of physical and biological agents during seed storage. To analyze the function of the testa in the model plant Arabidopsis, we compared mutants affected in testa pigmentation and/or structure for dormancy, germination, and storability. The seeds of most mutants exhibited reduced dormancy. Moreover, unlike wild-type testas, mutant testas were permeable to tetrazolium salts. These altered dormancy and tetrazolium uptake properties were related to defects in the pigmentation of the endothelium and its neighboring crushed parenchymatic layers, as determined by vanillin staining and microscopic observations. Structural aberrations such as missing layers or a modified epidermal layer in specific mutants also affected dormancy levels and permeability to tetrazolium. Both structural and pigmentation mutants deteriorated faster than the wild types during natural aging at room temperature, with structural mutants being the most strongly affected.     

Dormancy and Germination of Abscisic Acid-Deficient Tomato Seeds : Studies with the sitiens Mutant

The role of abscisic acid (ABA) in the dormancy induction of tomato (Lycopersicon esculentum) seeds was studied by comparison of the germination behavior of the ABA-deficient sitiens mutant with that of the isogenic wild-type genotype. Freshly harvested mutant seeds, in contrast to wild-type seeds, always readily germinate and even exhibit viviparous germination in overripe fruits. Crosses between mutant and wild-type and self-pollination of heterozygous plants show that in particular the ABA fraction of embryo and endosperm is decisive for the induction of dormancy. After-ripened wild-type seeds fully germinate in water but are more sensitive toward osmotic inhibition than mutant seeds. Germination of both wild-type and mutant seeds is equally sensitive toward inhibition by exogenous ABA. ABA content of mature wild-type seeds is about 10-fold the level found in mutant seeds. Nevertheless, it is argued that the differences in dormancy between the seeds of both genotypes are not a result of actual ABA levels in the mature seeds or fruits but a result of differences in ABA levels during seed development. It is hypothesized that the high levels of ABA that occur during seed development in wild-type seeds induce an inhibition of cell elongation of the radicle that can still be observed after long periods of dry storage.     

The regulator of G-protein signaling proteins involved in sugar and abscisic acid signaling in Arabidopsis seed germination

The regulator of G-protein signaling (RGS) proteins, recently identified in Arabidopsis (Arabidopsis thaliana; named as AtRGS1), has a predicted seven-transmembrane structure as well as an RGS box with GTPase-accelerating activity and thus desensitizes the G-protein-mediated signaling. The roles of AtRGS1 proteins in Arabidopsis seed germination and their possible interactions with sugars and abscisic acid (ABA) were investigated in this study. Using seeds that carry a null mutation in the genes encoding RGS protein (AtRGS1) and the alpha-subunit (AtGPA1) of the G protein in Arabidopsis (named rgs1-2 and gpa1-3, respectively), our genetic evidence proved the involvement of the AtRGS1 protein in the modulation of seed germination. In contrast to wild-type Columbia-0 and gpa1-3, stratification was found not to be required and the after-ripening process had no effect on the rgs1-2 seed germination. In addition, rgs1-2 seed germination was insensitive to glucose (Glc) and sucrose. The insensitivities of rgs1-2 to Glc and sucrose were not due to a possible osmotic stress because the germination of rgs1-2 mutant seeds showed the same response as those of gpa1-3 mutants and wild type when treated with the same concentrations of mannitol and sorbitol. The gpa1-3 seed germination was hypersensitive while rgs1-2 was less sensitive to exogenous ABA. The different responses to ABA largely diminished and the inhibitory effects on seed germination by exogenous ABA and Glc were markedly alleviated when endogenous ABA biosynthesis was inhibited. Hypersensitive responses of seed germination to both Glc and ABA were also observed in the overexpressor of AtRGS1. Analysis of the active endogenous ABA levels and the expression of NCED3 and ABA2 genes showed that Glc significantly stimulated the ABA biosynthesis and increased the expression of NCED3 and ABA2 genes in germinating Columbia seeds, but not in rgs1-2 mutant seeds. These data suggest that AtRGS1 proteins are involved in the regulation of seed germination. The hyposensitivity of rgs1-2 mutant seed germination to Glc might be the result of the impairment of ABA biosynthesis during seed germination.