NYEs/SGRs‐mediated chlorophyll degradation is critical for detoxification during seed maturation in Arabidopsis

In the seed industry, chlorophyll (Chl) fluorescence is often used as a major non‐invasive reporter of seed maturation and quality. Breakdown of Chl is a proactive process during the late stage of seed maturation, as well as during leaf senescence and fruit ripening. However, the biological significance of this process is still unclear. NYE1 and NYE2 are Mg‐dechelatases, catalyzing the first rate‐limiting step of Chl a degradation. Loss‐of‐function of both NYE1 and NYE2 not only results in a nearly complete retention of Chl during leaf senescence, but also produces green seeds in Arabidopsis. In this study, we showed that Chl retention in the nye1 nye2 double‐mutant caused severe photo‐damage to maturing seeds. Upon prolonged light exposure, green seeds of nye1 nye2 gradually bleached out and eventually lost their germination capacity. This organ‐specific photosensitive phenotype is likely due to an over‐accumulation of free Chl, which possesses photosensitizing properties and causes a burst of reactive oxygen species upon light exposure. As expected, a similar, albeit much milder, photosensitive phenotype was observed in the seeds of d1 d2, a green‐seed mutant defective in NYE/SGR orthologous genes in soybean. Taken together, our data suggest that efficient NYEs‐mediated Chl degradation is critical for detoxification during seed maturation.     

Relationship of chlorophyll, seed moisture and ABA levels in the maturing Brassica napus seed and effect of a mild freezing stress

Chlorophyll (Chl) retention by mature seed of canola as the result of an early frost or other environmental factors (the "green seed problem") causes serious economic losses. The relationship of seed degreening to rate of moisture loss by seed and silique and the role of ABA in this process were investigated as a function of developmental age. During the normal predesiccation stage (28–45 days after pollination), seed of Brassica napus (cv. Westar) loses Chl rapidly but seed moisture slowly. After a mild freezing stress, there is a rapid loss of moisture from silique walls, followed by accelerated loss of seed moisture. Chl degradation ceases at 35–45% seed moisture. ABA levels in silique walls of frozen plants (determined by enzyme‐linked immunosorbant assay) increased after freezing, apparently in response to moisture loss. In contrast, ABA levels in the seed increased dramatically 1 day after freezing, then decreased to control levels. The influence of the rate of seed moisture loss on Chl degradation was investigated by fast and slow drying of isolated seed under controlled humidity conditions. Seed dried rapidly at 22% RH retained most of its Chl, whereas seed dried slowly at 86% RH lost Chl as fast or faster than seed on control (unfrozen) plants. In all treatments, Chl loss stopped at about 40% seed moisture.     

Non-lethal freezing effects on seed degreening in Brassica napus

The effects of a non-lethal freezing stress on chlorophyll content, moisture level and distribution, and abscisic acid (ABA) levels were examined in siliques and seeds of Brassica napus (canola). A non-lethal freezing stress resulted in the retention of chlorophyll in seed at harvest that was most pronounced for seeds 28, 32 and 36 days after flowering (DAF). This increase was primarily due to an increased retention of chlorophyll a relative to chlorophyll b. Chlorophyll retention in seeds exposed to a non-lethal freezing stress correlated with an increased ABA catabolism, as measured 1, 3 or 7 days after the stress treatment. Although the non-lethal freezing stress had no significant effect on moisture content in seeds of siliques stressed at 28–44 DAF, moisture distribution, as viewed by magnetic resonance imaging, showed an uneven drying of 32 and 40 DAF siliques after exposure to the non-lethal freezing stress. Moisture was initially lost more rapidly from the silique wall between seeds, than in control non-stressed siliques. Increased moisture loss was not due to structural changes in the vasculature of the silique/seed of stressed tissues. These results are consistent with the hypothesis that a non-lethal freezing stress-induced decrease in ABA level, during seed maturation, effects an inhibition of normal chlorophyll a catabolism resulting in mature but green B. napus seed.     

Peroxidase-mediated chlorophyll bleaching in degreening canola (Brassica napus) seeds and its inhibition by sublethal freezing

Peroxidase activity in isolated thylakoids from degreening canola ( Brassica napus cv. Westar) seeds was demonstrated. The enzyme catalyzes the degradation of thylakoid-bound pigments in the presence of H₂O₂ and 2,4-dichlorophenol. Peroxidase activity is related to degreening, with periods of rapid degreening associated with high enzyme activity. Both de novo synthesis and substrate availability appear to control enzyme activity. Peroxidase is initially inhibited and then stimulated by sublethal freezing. Therefore, inhibition of peroxidase activity following sublethal freezing may be responsible, in part, for a failure of the seed to degreen.     

Chlorophyllase and peroxidase activity during degreening of maturing canola (Brassica napus) and mustard (Brassica juncea) seed

Chlorophyllase and peroxidase activities were measured in relation to seed maturation and degreening in canola ( Brassica napus cvs Westar and Alto) and mustard ( Brassica juncea cvs Cutlass and Lethbridge 22A). Samples of seed collected at the same moisture content were pooled, then divided and used for each assay. During maturation the green pigment (chlorophyll and related pigments) content of canola seed decreased linearly and was lower than that measured in mustard at all moisture contents studied, except for the highest and lowest moisture contents. Chlorophyllides and pheophorbides were detected in canola and were essentially absent in mustard. This difference in accumulation of dephytylated pigments infers differences in the pigment degradation pathways in Brassica species. Interspecific differences in the enzymology of degreening were found. Green pigment degradation was associated with increased chlorophyllase activity and low peroxidase activity in canola and low Chlorophyllase and high perosidase activity in mustard. The possible role of ethylene in seed degreening is discussed.     

Seed Vigour in Bean (Phaseolus vulgaris L. cv. Apollo) as Influenced by Temperature and Water Regime during Development and Maturation

Bean plants grown in a controlled temperature glasshouse athigh temperatures (33/28, 30/25, or 27/22 °C) during theperiod of seed development and maturation matured early andproduced small seeds. The seeds were of lower vigour than thosegrown at 21/16 or 18/13 °C. The detrimental effect of highmaturation temperatures was observed even on plants bearingwell-developed seeds (yellow, fleshy-pod stage). Seeds maturedat high temperatures were also more susceptible to deteriorationwith delay in harvest, and to mechanical damage. Heavy wateringof plants with seed ready to harvest caused a reduction in seedvigour. For optimum quality bean seed, it appears essentialthat the seed develops and matures at cool temperatures, ina dry environment.     

Accumulation and degradation of thiamin-binding protein and level of thiamin in wheat seeds during seed maturation and germination

Changes in the levels of thiamin-binding globulin and thiamin in wheat seeds during maturation and germination were studied. The thiamin-binding activity of the seed proteins increased with seed development after flowering. The thiamin content of the seeds also increased with development. Thiamin-binding activity decreased during seed germination. On the other hand, immunological analysis using an antibody directed against the thiamin-binding protein isolated from wheat seeds showed that the thiamin-binding globulin accumulated in the aleurone layer of the seeds during maturation, and then the protein was degraded and disappeared during seed germination. These results suggested that the thiamin-binding globulin of wheat seeds was synthesized and accumulated in the aleurone layer of the seeds with seed development, similar to the thiamin-binding albumin in sesame seeds, and that thiamin bound to the thiamin-binding globulin in the dormant wheat seeds for germ growth during germination.     

Protein Modification and Utilization of Starch in Soybean (Glycine max L. Merr.) Seed Maturation

Seeds of soybean (Glycine max L. Merr.) harvested at variousstages of development and allowed to dry in intact pods undergoa maturation process and are viable. Defatted powders of seedharvested 24–66 d after flowering were extracted to yieldbuffer-soluble and alkali-soluble proteins. Imposition of amaturation process increased the level of buffer-soluble proteinsbut had no effect on the disulflde content of these proteins.After undergoing maturation, seeds showed an accumulation ofbuffer-soluble polypeptides in the molecular weight range of43–94 kD. Maturation may be associated with the synthesisof specific polypeptides having a molecular weight of approximately85 kD. Alkali-soluble proteins, which represents the storageproteins, did not show any responses to maturation. Their quantityincreased substantially during seed development and the disulfidelevel was only half that of buffer-soluble proteins, attaininga maximum value of 10.9 mol S per 10⁵ g protein. Matured seedat all harvest dates had a final starch content close to thatof normal seed, 10–20 mg g^–1, and soluble sugarswere maintained at quite high levels, 51–83 mg g^–1.The metabolic program for synthesis and degradation of starchseems quite rigidly followed and is independent of harvest dateor of attachment to the parent plant. Soybean seeds retain considerablesoluble proteins and soluble sugars throughout maturation, andthese collectively may be important in maintaining a desiccationresistant structure.     

Quantification of ABA and its metabolites in sweet cherries usingdeuterium-labeled internal standards

Abscisic acid (ABA), phaseic acid (PA), dihydrophaseic acid (DPA), and epi-dihydrophaseic acid (epi-DPA) were quantified in developing fruit and seeds of sweet cherry using each deuterium-labeled internal standard. ABA concentrations in the pulp were low at the early stage of fruit development, reached to the maximum before maturation, and subsequently declined during maturation. The significant increase of ABA after 29 days after full bloom (DAFB) coincides with the softening suggests that ABA may play a role to induce fruit maturation in sweet cherries. ABA metabolite levels were high at the immature stage and decreased with fruit maturation. This fact suggests that fruit may not need ABA in the early stage of fruit development. It was considered that DPA may be the major metabolite of ABA since the concentrations were higher than PA and epi-DPA at all stages of fruit development. ABA concentrations increased at the beginning of seed maturation and then decreased toward harvest. This decrease may be necessary to end seed dormancy. DPA in seeds changed similarly with ABA but its concentrations were always higher than those of ABA.     

Sugar metabolism in developing lupin seeds is affected by a short-term water deficit

A short-term water deficit (WD) imposed during the pre-storage phase of lupin seed development [15-22 d after anthesis (DAA)] accelerated seed maturation and led to smaller and lighter seeds. During seed development, neutral invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) have a central role in carbohydrate metabolism. Neutral invertase is predominant during early seed development (up to 40 DAA) and sucrose synthase during the growing and storage phase (40-70 DAA). The contribution of acid invertase is marginal. WD decreased sucrose synthase activity by 2-fold and neutral invertase activity by 5-6-fold. These changes were linked to a large decrease in sucrose ( approximately 60%) and an increase of the hexose:sucrose ratio. Rewatering restored sucrose synthase activity to control levels while neutral invertase activity remained depressed (30-60%). A transient accumulation of starch observed in control seeds was abolished by WD. Despite the several metabolic changes the final seed composition was largely unaltered by WD except for approximately 60% increase in stachyose and raffinose (raffinose family oligosaccharides). This increase in raffinose family oligosaccharides appears as the WD imprinting on mature seeds.     

Protein Synthesis During Rehydration, Germination and Seedling Growth of Naturally and Precociously Matured Soybean Seeds (Glycine max)

Soybean seeds [Glycine max (L.) Merr.] synthesize de novo andaccumulate several non-storage, soluble polypeptides duringnatural and precocious seed maturation. These polypeptides havepreviously been coined ‘maturation polypeptides’.The objective of this study was to determine the fate of maturationpolypeptides in naturally and precociously matured soybean seedsduring rehydration, germination, and seedling growth. Developingsoybean seeds harvested 35 d after flowering (mid-development)were precociously matured through controlled dehydration, whereasnaturally matured soybean seeds were harvested directly fromthe plant. Seeds were rehydrated with water for various timesbetween 5 and 120 h. Total soluble proteins and proteins radio-labelledin vivo were extracted from the cotyledons and embryonic axesof precociously and naturally matured and rehydrated seed tissuesand analyzed by one-dimensional PAGE and fluorography. The resultsindicated that three of the maturation polypeptides (21, 31and 128 kDa) that had accumulated in the maturing seeds (maturationpolypeptides) continued to be synthesized during early stagesof seed rehydration and germination (5–30 h after imbibition).However, the progression from seed germination into seedlinggrowth (between 30 and 72 h after imbibition) was marked bythe cessation of synthesis of the maturation polypeptides followedby the hydrolysis of storage polypeptides that had been synthesizedand accumulated during seed development. This implied a drasticredirection in seed metabolism for the precociously maturedseeds as these seeds, if not matured early, would have continuedto synthesize storage protein reserves. Glycine max (L.) Merr, soybean, cotyledons, maturation, germination/seedling growth     

Cytochemical Localization of Phenolic Compounds in Columella Cells of the Root Cap during Maturation of Seeds of Brassica napus L.

Abstract: Ultrastructural localization of phenolic compounds in the columella of the root cap of a canola embryo in Brassica napus L. during subsequent seed maturation stages was investigated. In bright green seeds the first signs of maturation and appearance of the first phenolic structures in the cell nuclei and cytoplasm in the area of the initial centre were found. Phenolics spread acropetally, gradually covering the whole columella in the dark green seeds. An increase in the number of phenolics occurred, together with an increase in the number of endoplasmic reticulum structures and in storage materials. In brown seeds, together with ultrastructural degradation and the end of production of storage substances, the number of phenolic deposits in the nucleus and cytoplasm decreases until their disappearance in black dormant seeds. Simultaneously, numerous phenolic deposits appear between the plasmalemma and the cell walls in the outer layer of the cap columella.     

Phosphoenolpyruvate Carboxylase during Maturation and Germination Sorghum Seeds: Enzyme Activity and Regulation

Sorghum (Sorghum bicolor (L.) Moench) is a species of great socio-economic and ecological importance for countries in arid and semi-arid climate. In C₄ plants like sorghum, phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) plays a key role in seed development and germination. In this work, the PEPC activity shows an increase followed by a decrease at the early and later stages of maturation, respectively. In germinating seeds, the PEPC activity quickly increases after soaking. The L-malate test and the ratio of PEPC activity determined at pH 8.0 and 7.1, indicates, that PEPC is phosphorylated at the early stages of maturation then becomes dephosphorylated at the later stages and during seed germination, PEPC takes back its phosphorylated form. The determination of the affinity constant showed different K_M depending on the seed developmental stage. As there is no PEPC-C4 isoform in developing sorghum seeds, this result indicates that the different K_M observed during seed maturation could be a result of a post-translational regulation such as phosphorylation or ubiquitination of a pre-existing isoform. This regulation enhances the PEPC activity at early stages of seed development.     

Ethylene Production during Development of Mustard (Brassica juncea) and Canola (Brassica napus) Seed

An open, continuous flow system was used to investigate ethylene production during degreening of maturing seed of mustard (Brassica juncea cv Cutlass and cv Lethbridge 22A) and canola (Brassica napus cv Westar and cv Alto). Isolated mustard seed evolved higher amounts of ethylene than those of canola, and this was particularly evident both early in embryogeny and later during the desiccation phase of seed maturation. The silique walls produced negligible amounts of ethylene in both species. The concentrations of ethylene surrounding seed as they matured within siliques were significantly higher in mustard than in canola, and this interspecies difference was greatest during the seed desiccation phase. In mustard, a 4-fold increase in silique internal ethylene levels was apparent during desiccation. In comparison, only a moderate increase in silique-derived ethylene occurred in canola.     

Asparaginyl endopeptidase during maturation and germination of durum wheat

Asparaginyl-endopeptidase activity was detected in endosperms of maturing and germinating wheat seeds. The highest activity was found during maturation before the maximal accumulation of storage proteins. The enzyme activity then decreased in the dry seeds and increased again during germination. The increase of activity during germination required the presence of the embryo. In fact, the activity found in detached endosperms was lower than that found in attached ones. The localization at tissue level of the enzyme reveals differences between maturation and germination: the enzyme was about equally located in the aleurone layer and starchy endosperm during maturation, but solely in the aleurone layer during germination. The asparaginyl enzymes from maturing and germinating seeds had many similar properties, such as pH optimum, pH stability, thermal stability and sensitivity to thiol reagents and to thiol compounds. The results suggest that asparaginyl endopeptidases may be involved in the modification of proproteins of storage proteins during seed maturation and in the degradation of storage proteins deposited in the aleurone layer during germination.     

Change of thiamin-binding protein and thiamin levels during seed maturation and germination in sesame

Thiamin-binding proteins (TBPs) occur in many types of plant seeds. The biochemical and structural properties such as subunit structure and affinity for thiamin of the proteins have been characterized. However, the change of TBP and thiamin during seed maturation and germination is little known. Sesame (Sesamum indicum L.) seeds have unique albumin TBPs, because the other TBPs from plant seeds are generally globulins. In this study, we studied the change of the TBP and thiamin levels in sesame seeds. The protein content and thiamin-binding activity of the seeds increased with seed development after flowering. Immunological analysis using an antibody against the TBP of sesame seeds showed that the protein was accumulated in seeds during maturation. The thiamin content of the seeds increased with seed development after flowering. On the other hand, the thiamin-binding activity decreased during seed germination when TBP was degraded. The thiamin content of the seeds decreased during the germination. However, the amount of thiamin phosphate in the seeds during germination was little changed. These results suggested that thiamin was accumulated and stored as a complex with TBP in sesame seeds.     

Influence of honey bee (Hymenoptera: Apidae) density on the production of canola (Crucifera: Brassicacae)

Pollination is an essential step in the seed production of canola, Brassica napus L. It is achieved with the assistance of various pollen vectors, but particularly by the honey bee, Apis mellifera L. Although the importance of pollination has been shown for the production of seed crops, the need to introduce bee hives in canola fields during flowering to increase oil seed yield has not yet been proven. With the purpose of showing this, hives of A. mellifera were grouped and placed in various canola fields in the Chaudière-Appalaches and Capitale-Nationale regions (nine fields; three blocks with three treatments; 0, 1.5, and 3 hives per hectare). A cage was used to exclude pollinators and bee visitations were observed in each field. After the harvest, yield analyses were done in relation to the bee density gradient created, by using pod set, number of seeds per plant, and weight of 1000 seeds. Results showed an improvement in seed yield of 46% in the presence of three honey bee hives per hectare, compared with the absence of hives. The introduction of honey bees contributed to production and consequently, these pollinators represented a beneficial and important pollen vector for the optimal yield of canola.     

Abscisic Acid and Gibberellic Acid Contents in Ripening Barley Seeds

The ABA and GA3 contents were investigated in barley seeds during maturation and after harvest. The highest amount of ABA was found in milk and wax ripeness – 13 ng and 11 ng per seed respectively. The level decreased during the later stages of maturation and during release of dormancy and was 1 ng per seed 6 weeks after harvest. The content of gibberellic acid decreased in a similar way but in an earlier stage of maturation. The determined amounts of GA₃ were: 0.4, 0.1, 0.03 and about 0.05 ng per seed respectively, in milk, wax and full ripeness and after harvest. The results of quantitative determination, obtained with the GLC method, corresponded to the growth effects in the test of the first leaf of oat.     

Molecular cloning and function analysis of the stay green gene in rice

Chloroplasts undergo drastic morphological and physiological changes during senescence with a visible symptom of chlorophyll (Chl) degradation. A stay green mutant was identified and then isolated from the japonica rice (Oryza sativa) cv. Huazhiwu by gamma-ray irradiation. The stay green mutant was characterized by Chl retention, stable Chl-protein complexes, and stable thylakoid membrane structures, but lost its photosynthetic competence during senescence. The gene, designated Stay Green Rice (SGR), was cloned by a positional cloning strategy encoding an ancient protein containing a putative chloroplast transit peptide. SGR protein was found in both soluble and thylakoid membranes in rice. SGR, like the gene for pheophorbide a oxygenase (PaO), was constitutively expressed, but was upregulated by dark-induced senescence in rice leaves. Senescence-induced expression of SGR and PaO was enhanced by ABA, but inhibited by cytokinin. Overexpression of SGR reduced the number of lamellae in the grana thylakoids and reduced the Chl content of normally growing leaves. This indicates that upregulation of SGR increases Chl breakdown during senescence in rice. A small quantity of chlorophyllide a accumulated in sgr leaves, but this also accumulated in wild-type rice leaves during senescence. Some pheophorbide a was detected in sgr leaves in the dark. According to these observations, we propose that SGR may be involved in regulating or taking part in the activity of PaO, and then may influence Chl breakdown and degradation of pigment-protein complex.