Changes in RNA and Protein Metabolism Associated with Alterations in the Germination Efficiency of Sunflower Seeds

Precise knowledge of seed quality after harvest and during storageis of particular importance for seed producers. We analyseddifferent sunflower seed lots (Helianthus annuusL.) characterizedby extremes of germination ability. We used RNA analysis tostudy possible changes in gene expression in seeds unable togerminate. Total RNA content was very small in dry seeds showinga low germination ability. Capacity for total RNA synthesisat the onset of imbibition was also reduced in these seeds.In addition, correlations were found between these parametersand germination ability at 19 °C. We demonstrated a highcorrelation between the amount of total RNA in the dry seed,the capacity of RNA synthesis at the onset of imbibition andthe seed moisture content at the time of the harvest. The abilityof dry seed mRNAs to be translatedin vitrowas also reduced andseven polypeptides, from stored mRNAs, were characteristic ofthe cotyledons from high germinability seeds. Germination canthus be affected at several levels including membrane, enzymaticand nucleic acid deteriorations. Gene expression; germination ability; Helianthus annuusL.; marker; protein; RNA; seed; sunflower     

Proteomic analysis of seed dormancy in Arabidopsis

The mechanisms controlling seed dormancy in Arabidopsis (Arabidopsis thaliana) have been characterized by proteomics using the dormant (D) accession Cvi originating from the Cape Verde Islands. Comparative studies carried out with freshly harvested dormant and after-ripened non-dormant (ND) seeds revealed a specific differential accumulation of 32 proteins. The data suggested that proteins associated with metabolic functions potentially involved in germination can accumulate during after-ripening in the dry state leading to dormancy release. Exogenous application of abscisic acid (ABA) to ND seeds strongly impeded their germination, which physiologically mimicked the behavior of D imbibed seeds. This application resulted in an alteration of the accumulation pattern of 71 proteins. There was a strong down-accumulation of a major part (90%) of these proteins, which were involved mainly in energetic and protein metabolisms. This feature suggested that exogenous ABA triggers proteolytic mechanisms in imbibed seeds. An analysis of de novo protein synthesis by two-dimensional gel electrophoresis in the presence of [(35)S]-methionine disclosed that exogenous ABA does not impede protein biosynthesis during imbibition. Furthermore, imbibed D seeds proved competent for de novo protein synthesis, demonstrating that impediment of protein translation was not the cause of the observed block of seed germination. However, the two-dimensional protein profiles were markedly different from those obtained with the ND seeds imbibed in ABA. Altogether, the data showed that the mechanisms blocking germination of the ND seeds by ABA application are different from those preventing germination of the D seeds imbibed in basal medium.     

Changes in endogenous abscisic acid levels during dormancy release and maintenance of mature seeds: studies with the Cape Verde Islands ecotype,the dormant model of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Mature seeds of the Cape Verde Islands (Cvi) ecotype of Arabidopsis thaliana (L.) Heynh. show a very marked dormancy. Dormant (D) seeds completely fail to germinate in conditions that are favourable for germination whereas non-dormant (ND) seeds germinate easily. Cvi seed dormancy is alleviated by after-ripening, stratification, and also by nitrate or fluridone treatment. Addition of gibberellins to D seeds does not suppress dormancy efficiently, suggesting that gibberellins are not directly involved in the breaking of dormancy. Dormancy expression of Cvi seeds is strongly dependent on temperature: D seeds do not germinate at warm temperatures (20–27°C) but do so easily at a low temperature (13°C) or when a fluridone treatment is given to D seeds sown at high temperature. To investigate the role of abscisic acid (ABA) in dormancy release and maintenance, we measured the ABA content in both ND and D seeds imbibed using various dormancy-breaking conditions. It was found that dry D seeds contained higher amounts of ABA than dry ND after-ripened seeds. During early imbibition in standard conditions, there was a decrease in ABA content in both seeds, the rate of which was slower in D seeds. Three days after sowing, the ABA content in D seeds increased specifically and then remained at a high level. When imbibed with fluridone, nitrate or stratified, the ABA content of D seeds decreased and reached a level very near to that of ND seeds. In contrast, gibberellic acid (GA₃) treatment caused a transient increase in ABA content. When D seeds were sown at low optimal temperature their ABA content also decreased to the level observed in ND seeds. The present study indicates that Cvi D and ND seeds can be easily distinguished by their ability to synthesize ABA following imbibition. Treatments used here to break dormancy reduced the ABA level in imbibed D seeds to the level observed in ND seeds, with the exception of GA₃ treatment, which was active in promoting germination only when ABA synthesis was inhibited.Abbreviations ABA Abscisic acid - Cvi Cape Verde Islands - D Dormant - GA Gibberellin - GA₃ Gibberellic acid - ND Non dormant     

Spatially regulated genes expressed during seed germination and postgerminative development are activated during embryogeny

Summary We investigated the control of genes expressed primarily during seed germination and postgerminative development in Brassica napus L. We identified cloned mRNA sequences which became prevalent within 1 day after the start of imbibition and were at low or undetectable levels in immature embryos, dry seeds, and leaves. Most postgermination-abundant mRNAs accumulated primarily, though not exclusively, in different parts of the seedling. Of the 14 cloned mRNAs, 8 were prevalent in cotyledons, 2 were abundant in seedling axes, and 4 were approximately equally distributed in both parts. We showed that although these mRNAs reached maximal levels in seedlings, the spatially regulated mRNAs were also detected at distinct embryonic stages; mRNAs prevalent in seedling axes accumulated primarily during early embryogenesis while cotyledon-abundant mRNA concentration increased during late embryogeny. We conclude that the temporal and spatial regulation of gene expression in seedlings reflects similarities and differences in the physiological functions of cotyledons and axes. Furthermore, the regulated expression of cotyledon-abundant genes during late embryogeny suggests that the mRNAs and possibly proteins may accumulate in preparation for subsequent seedling growth. Similarities in the accumulation of cotyledon-abundant mRNAs may indicate coordinate regulation of this gene set.Abbreviations DAF days after flowering - DAI days after the start of imbibition - HAI hours after the start of imbibition - kb kilobase(pairs)     

Comparison of globulin mobilization and cysteine proteinases in embryonic axes and cotyledons during germination and seedling growth of vetch (Vicia sativa L.)

Vicilin and legumin, the storage globulins of mature dry vetch (Vicia sativa L.) seeds, are found in protein bodies which are present not only in the cotyledons, but also in the radicle, axis and shoot (together, for reasons of simplicity, here called axis). When at 24 h after the start of imbibition (hai) the radicle breaks through the seed coat a major part of the globulins in the axis has already been degraded, whereas in the cotyledons globulin breakdown cannot yet be detected. Globulin mobilization starts with the degradation of vicilin. At 48 hai when globulin mobilization in the cotyledons just begins, the axis is already nearly depleted of globulins. Mobilization of storage globulin is probably brought about by a complex of different cysteine proteinases (CPRs). The papain-like CPR2 and CPR4, and the legumain-like VsPB2, together with their mRNAs, are already present in axes and cotyledons of dry seeds. This means that they must have been formed during seed maturation. Additional papain-like CPRs are formed later during germination and seedling growth. CPR4 and VsPB2 together with their corresponding mRNAs become undetectable as germination and seedling growth proceed. VsPB2 and VsPB2-mRNA are substituted by the homologous legumain-like proteinase B and its mRNA. The composition of stored and newly formed CPRs undergoes developmental changes which differ between axes and cotyledons. It is concluded that storage globulin mobilization in germinating vetch seeds is started by stored CPRs, whereas the mobilization of the bulk of globulin is predominantly mediated by CPRs which are formed de novo.     

Stored proteinases and the initiation of storage protein mobilization in seeds during germination and seedling growth

Though endopeptidases and carboxypeptidases are present in protein bodies of dry quiescent seeds the function of these proteases during germination is still a matter of debate. In some plants it was demonstrated that endopeptidases of dry protein bodies degrade storage proteins of these organelles. Other studies describe cases where this did not happen. The role that stored proteinases play in the initiation of storage protein breakdown in germinating seeds thus remains unclear. Numerous reviews state that the initiation of reserve protein mobilization is attributed to de novo formed endopeptidases which together with stored carboxypeptidases degrade the bulk of proteins in storage organs and tissues after seeds have germinated. The evidence that the small amounts of endopeptidases in protein bodies of embryonic axes and cotyledons of dry seeds from dicotyledonous plants play an important role in the initiation of storage protein mobilization during early germination is summarized here.     

Wheat cysteine proteases triticain alpha, beta and gamma exhibit mutually distinct responses to gibberellin in germinating seeds

We cloned three novel papain-type cysteine proteases (CPs), triticain alpha, beta and gamma, from 1-d-germinating wheat seeds. Triticain alpha, beta and gamma were constituted with 461, 472 and 365 amino acid residues, respectively, and had Cys-His-Asn catalytic triads as well as signal and propeptide sequences. Triticain gamma contained a putative vacuole-sorting sequence. Phylogenetic analysis showed that these CPs were divided into mutually different clusters. Triticain alpha and gamma mRNAs were expressed in seeds at an early stage of maturation and at the stage of germination 2d after imbibition, while triticain beta mRNA appeared shortly after imbibition. The expression of mRNAs for triticain alpha and gamma was suppressed by uniconazol, a gibberellin synthesis inhibitor. All the three CP mRNAs were strongly expressed in both embryo and aleurone layers. These results suggest that triticain alpha, beta and gamma play differential roles in seed maturation as well as in digestion of storage proteins during germination.     

The nuclear protein Poly(ADP‐ribose) polymerase 3 (AtPARP3) is required for seed storability in Arabidopsis thaliana

The deterioration of seeds during prolonged storage results in a reduction of viability and germination rate. DNA damage is one of the major cellular defects associated with seed deterioration. It is provoked by the formation of reactive oxygen species (ROS) even in the quiescent state of the desiccated seed. In contrast to other stages of seed life, DNA repair during storage is hindered through the low seed water content; thereby DNA lesions can accumulate. To allow subsequent seedling development, DNA repair has thus to be initiated immediately upon imbibition. Poly(ADP‐ribose) polymerases (PARPs) are important components in the DNA damage response in humans. Arabidopsis thaliana contains three homologues to the human HsPARP1 protein. Of these three, only AtPARP3 was very highly expressed in seeds. Histochemical GUS staining of embryos and endosperm layers revealed strong promoter activity of AtPARP3 during all steps of germination. This coincided with high ROS activity and indicated a role of the nuclear‐localised AtPARP3 in DNA repair during germination. Accordingly, stored parp3‐1 mutant seeds lacking AtPARP3 expression displayed a delay in germination as compared to Col‐0 wild‐type seeds. A controlled deterioration test showed that the mutant seeds were hypersensitive to unfavourable storage conditions. The results demonstrate that AtPARP3 is an important component of seed storability and viability.     

Exudation of an allelopathic substance lepidimoide from seeds during germination

Lepidimoide promotes growth of the cockscomb hypocotyl. It is exuded from germinating seeds of various plant species into their culture environment. The mode of exudation of lepidimoide from seeds into the culture solution, using sunflower and buckwheat seeds, was studied in relation to seed germination. In the dry seeds, about 75% of the lepidimoide was found in the seed coat and about 25% in the kernel. Upon water imbibition it was released into the culture solution. However, the quantity of lepidimoide detected in the seed exudate was more than three times the total amount in dry and imbibed seeds, suggesting that lepidimoide was also produced de novo in the seeds and subsequently released. When seed coats or kernels were imbibed separately, the quantity of lepidimoide released from the seed coats into the culture solution was much the same as that in the dry seeds, but the amount of lepidimoide detected in the exudate of kernels was about 16 times that in the dry kernels. These results suggest that lepidimoide, already present in dry seeds, as well as that newly produced in the kernels following imbibition, was released into the environment.