Toward characterizing seed vigor in alfalfa through proteomic analysis of germination and priming

Alfalfa, the most widely grown leguminous crop in the world, is generally exposed to severe salinity stress in Tunisia, notably affecting its germination performance. Toward a better understanding of alfalfa seed vigor, we have used proteomics to characterize protein changes occurring during germination and osmopriming, a pretreatment that accelerates germination and improves seedling uniformity particularly under stress conditions. The data revealed that germination was accompanied by dynamic changes of 79 proteins, which are mainly involved in protein metabolism, cell structure, metabolism, and defense. Comparative proteomic analysis also revealed 63 proteins specific to osmopriming, 65 proteins preferentially varying during germination, and 14 proteins common to both conditions. Thus, the present study unveiled the unexpected finding that osmopriming cannot simply be considered as an advance of germination-related processes but involves other mechanisms improving germination such as the mounting of defense mechanisms enabling osmoprimed seeds to surmount environmental stresses potentially occurring during germination. The present results therefore provide novel avenues toward understanding the mechanisms of invigoration of low vigor seeds by priming treatments that are widely used both in commercial applications and in developing countries (on farm seed priming) to better control crop yields.     

Aquaporin expression during seed osmopriming and post-priming germination in spinach

Aquaporins (AQPs) are proteinaceous channels known to regulate transmembrane water transport, and therefore may be important component of imbibition during osmopriming and germination. To explore the association between AQPs and osmopriming-led enhanced germination performance, we studied the expression patterns of four spinach (Spinacia oleracea) AQP coding genes (SoPIP1;1, SoPIP1;2, SoPIP2;1, and SoδTIP) during osmopriming and subsequent germination under optimal conditions, chilling and drought. All these genes were up-regulated within 2–4 d of priming (phase II-imbibition). We hypothesize such up-regulation to facilitate the pressure potential-driven cell expansion and increase germination potential of primed seeds. Our data during post-priming germination suggest that SoPIP1;1 and SoδTIP were more closely associated with enhanced germination performance. In general, all AQPs were downregulated under chilling and drought. However, under chilling, SoPIP2;1 was expressed at relatively higher level in primed seeds that also exhibited greater chilling tolerance, while SoPIP1;2 and SoδTIP exhibited opposite pattern. Similarly, SoPIP1;1, SoPIP2;1, and SoδTIP exhibited higher expression in primed seeds that also had greater drought tolerance.     

Selection of Reference Genes for Normalizing Gene Expression During Seed Priming and Germination Using qPCR in Zea mays and Spinacia oleracea

Quantitative real-time RT-PCR (qPCR) has been widely used to investigate gene expression during seed germination, a process involving seed transition from dry/physiologically inactive to hydrated/active state. This transition may result in altered expression of many housekeeping genes (HKGs), conventionally used as internal controls, thereby posing a challenge about selection of HKGs in such scenarios. The objectives of this study included identifying valid reference genes for seed priming and germination studies, both of which involve the transition of seed hydration status, and assessing whether or not findings derived from the “seed model” used in this study would also be applicable to other plant species. Eight commonly used HKGs were evaluated in maize seeds during hydropriming and germination. Using Bestkeeper, geNorm, and NormFinder, we provided a rank of stability for these HKGs. Actdf, UBQ, βtub, 18S, Act, and GAPDH were adjudged as valid internal controls by geNorm and NormFinder. Under the second objective, we conducted a case study with spinach seeds collected during osmopriming and germination. Our results indicate that the conclusions derived from maize were applicable to spinach as well, in that 18S exhibited greater expression stability than GAPDH in osmoprimed and germinated seeds; this held true even under stress conditions. While both of these genes were rejected by BestKeeper, we found that 18S exhibited stable expression when “dry” and “hydrated” seeds were analyzed as separate data sets. Although this approach precludes the comparison between “hydrated” and “dry” seeds, it still provides effective comparison among samples of same hydration status.     

Drought tolerance of Periploca sepium during seed germination: antioxidant defense and compatible solutes accumulation

Periploca sepium Bunge is a native and widespread shrub on the Loess Plateau, an arid and semi-arid region in China. To understand the adaptability of its seed germination to dry environments, we investigated the germination rate, water relations, lipid peroxidation, antioxidant capacity and accumulation of major organic solutes during seed germination under water deficit conditions. Results showed that seeds pre-treated by hydration–dehydration or ?0.9 MPa PEG germinated faster than control seeds, indicating strong resistance of P. sepium to drought condition. The re-dried seeds showed higher proline, total free amino acids (TFAA) and soluble proteins (SP) contents than control dry seeds, indicating the maintenance of physiological advancement when dehydrated. Osmotic stress made seed germination stay on the plateau phase (phase II). However, germinating seeds moved into phase III immediately once transferred into distilled water. Large increases in SP and soluble sugars (SS) of both re-dried and osmotic stressed seeds help themselves to resist drought stress. The re-hydrated seeds showed significantly higher levels of proline, TFAA, SP and SS than control seeds. The largely accumulated SS during osmotic stress declined sharply when transferred into distilled water. Our data demonstrate that P. sepium’s tolerance to drought stresses during germination is associated with enhanced activity of antioxidant enzymes and accumulation of some compatible solutes. Seed physiological advancement progressed slowly under low water conditions and it was maintained when seeds were air dried. This strategy ensures high and more rapid seed germination of P. sepium under drying and wetting conditions in drought-prone regions.     

Polyethylene glycol 6000 priming effect on germination of aged wheat seed lots

The effects of PEG 6000 priming on germination performance of aged wheat seed lots have been studied. A correct application of osmopriming treatment indicated a relationship between the pattern of water absorption, the reactivation of mitotic activity and the start and synchronization of germination. The possibility of controlling pregerminative events by means of this treatment is discussed on the physiological basis of seed germination.     

Repressing the expression of the SUCROSE NONFERMENTING-1-RELATED PROTEIN KINASE gene in pea embryo causes pleiotropic defects of maturation similar to an abscisic acid-insensitive phenotype

The classic role of SUCROSE NONFERMENTING-1 (Snf1)-like kinases in eukaryotes is to adapt metabolism to environmental conditions such as nutrition, energy, and stress. During pea (Pisum sativum) seed maturation, developmental programs of growing embryos are adjusted to changing physiological and metabolic conditions. To understand regulation of the switch from cell proliferation to differentiation, SUCROSE NONFERMENTING-1-RELATED PROTEIN KINASE (SnRK1) was antisense repressed in pea seeds. Transgenic seeds show maturation defects, reduced conversion of sucrose into storage products, lower globulin content, frequently altered cotyledon surface, shape, and symmetry, as well as occasional precocious germination. Gene expression analysis of embryos using macroarrays of 5,548 seed-specific genes revealed 183 differentially expressed genes in two clusters, either delayed down-regulated or delayed up-regulated during transition. Delayed down-regulated genes are related to mitotic activity, gibberellic acid/brassinosteroid synthesis, stress response, and Ca2+ signal transduction. This specifies a developmentally younger status and conditional stress. Higher gene expression related to respiration/gluconeogenesis/fermentation is consistent with a role of SnRK1 in repressing energy-consuming processes in maturing cotyledons under low oxygen/energy availability. Delayed up-regulated genes are mainly related to storage protein synthesis and stress tolerance. Most of the phenotype resembles abscisic acid (ABA) insensitivity and may be explained by reduced Abi-3 expression. This may cause a reduction in ABA functions and/or a disconnection between metabolic and ABA signals, suggesting that SnRK1 is a mediator of ABA functions during pea seed maturation. SnRK1 repression also impairs gene expression associated with differentiation, independent from ABA functions, like regulation and signaling of developmental events, chromatin reorganization, cell wall synthesis, biosynthetic activity of plastids, and regulated proteolysis.     

The Effect of Chilling and Moisture Status on the Germination, Desiccation Tolerance and Longevity ofAesculus hippocastanumL. Seed

Effects of 2 °C chilling on the threshold moisture contentsand water potentials for various physiological processes wereestimated forAesculus hippocastanumL. seed. Seed harvested atthe time of maximum seed fall exhibited a dual response to drying:partial drying from near 50% to 32–40% moisture contentprogressively increased germination percentage (at 16 °C)up to various peak values; further desiccation was detrimental,confirming that the seeds are ‘recalcitrant’. Themoisture content for optimum germination was increased by atleast 10% as the chilling period was raised from 0 to 9 weeks.A negative linear relationship was found between log₁₀mean timeto germinate and probit final germination, regardless of pre-treatment,indicating that partial desiccation and chilling are interchangeablein promoting germination of hydrated seed. For nearly fullyhydrated seeds, increasing the chilling period from 6 to 26weeks increased the viability-loss onset point for desiccationinjury from near 40% to about 48% moisture content without alteringthe drying rates of seed tissues. Extending moist chilling invarious seed lots from 0 to 26 weeks decreased subsequent longevityat 16 °C. For 26-week-chilled seeds longevity (the periodto lose one probit of germination) differed above and belowa threshold moisture content of 48%. It remained constant inthe moisture-content range 48–38%, but increased progressivelyas moisture content was raised above 48%. This threshold moisturecontent coincided with the value above which chilled seed pre-germinatedin storage. The results indicate that post-harvest desiccationand chilling alter the water relations of various physiologicalprocesses and a schematic summary is presented which relatesthe results to an axis water sorption isotherm.Copyright 1998Annals of Botany Company Aesculus hippocastanumL., horse chestnut, chilling, moisture content, water potential, desiccation tolerance, longevity, recalcitrant seed, embryo axis, maturation, germination.     

Effect of stimulating maize germination on cell cycle proteins

The germination process can be accelerated if seeds are stimulated either by adding cytokinins or by osmopriming. Under these conditions, cells in maize ( Zea mays ) embryo axes shorten the time at which the first round of DNA replication and mitosis takes place, thus advancing the cell cycle. Using heterologous antibodies against different cell cycle proteins, we have followed the behaviour of several markers for G1 phase (cyclin D, E2F and p53) and a marker of G2 phase (cyclin B) under either control or "accelerated" germination conditions. The results showed two classes of behaviour: either there was no variation in the amount of the protein present under control or accelerated germination conditions, represented by cyclin Band E2F‐type proteins, or the amount of the proteins was drastically reduced, more rapidly under accelerated germination, as was the case for cyclin D‐ and p53‐type proteins. Although the cyclin D‐type protein was synthesized de novo during germination, the balance was towards degradation so that there was no cyclin D detected 15 h after germination in benzyladenine‐treated and osmoprimed seeds. A Cdk4‐type protein seemed to be present in cyclin D immunoprecipitates and its kinase activity paralleled the fluctuations of the cyclin amount during germination. These data are discussed in the context of early seed germination.     

A wheat homolog of MOTHER OF FT AND TFL1 acts in the regulation of germination

Seed dormancy is an adaptive mechanism and an important agronomic trait. Temperature during seed development strongly affects seed dormancy in wheat (Triticum aestivum) with lower temperatures producing higher levels of seed dormancy. To identify genes important for seed dormancy, we used a wheat microarray to analyze gene expression in embryos from mature seeds grown at lower and higher temperatures. We found that a wheat homolog of MOTHER OF FT AND TFL1 (MFT) was upregulated after physiological maturity in dormant seeds grown at the lower temperature. In situ hybridization analysis indicated that MFT was exclusively expressed in the scutellum and coleorhiza. Mapping analysis showed that MFT on chromosome 3A (MFT-3A) colocalized with the seed dormancy quantitative trait locus (QTL) QPhs.ocs-3A.1. MFT-3A expression levels in a dormant cultivar used for the detection of the QTL were higher after physiological maturity; this increased expression correlated with a single nucleotide polymorphism in the promoter region. In a complementation analysis, high levels of MFT expression were correlated with a low germination index in T1 seeds. Furthermore, precocious germination of isolated immature embryos was suppressed by transient introduction of MFT driven by the maize (Zea mays) ubiquitin promoter. Taken together, these results suggest that MFT plays an important role in the regulation of germination in wheat.     

Expression of three ABA-regulated clones and their relationship to maturation processes during the embryogenesis of chick-pea seeds

Water stress inhibits germination of chick-pea seeds and produces specific changes in gene expression. some of which are coincident with those induced by the exogenous application of abscisic acid (ABA). Three cDNA clones, GAB-8, GAB-9 and GAB-11, were previously identified as under the regulation of ABA and osmotic stress in embryonic axes of germinating chick-pea. Here we try to establish a relationship between the changes in gene expression induced by ABA and stress conditions during germination and those naturally occurring during the desiccation process that leads to seed maturation. Our results show that the germinative capacity of chick-pea is related to the water content of the organ. In vitro translation of the mRNAs from developing seed reveals that in the later stages of seed maturation some polypeptides appear that previously were found to be regulated by ABA and by water deficit in germinating seeds. Hybridization by northern blot of embryogenic mRNAs with GAB-8. GAB-9 and GAB-11 clones shows that the mRNAs corresponding to such clones only appear in the later phases of seed formation, coinciding with seed dehydration, and persisting until seeds became fully mature. The results suggest that these mRNAs are probably related to the response to dehydration that occurs during seed maturation, and that the pattern of expression of these ABA-regulated clones coincides with that of the established late embryogenesis-abundant (LEA) genes.     

Preliminary Study on Development,Germination and Desiccation Tolerance of Jatropha curcas (Euphorbiaceae)

The developmental changes in morphology and germinability of Jatropha curcas seeds, effects of temperature and light on seed germination , and changes in desiccation tolerance of mature seeds were studied in this paper. The results indicated that J. curcas seeds reached a physiological maturation at 58 days after anthesis, and that seed germination percentage reached a peak at physiological maturation, and then decreased. The optimal germination temperature was 25 to 30℃. J. curcas seeds were insensitive to dehydration at physiological maturation . There was not a notable effect of light on seed germination. Therefore, J. curcas seed was a non-photoblastic and orthodox seeds .     

In vivo analysis of metal distribution and expression of metal transporters in rice seed during germination process by microarray and X-ray Fluorescence Imaging of Fe, Zn, Mn, and Cu

To investigate the flow of the metal nutrients iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) during rice seed germination, we performed microarray analysis to examine the expression of genes involved in metal transport. Many kinds of metal transporter genes were strongly expressed and their expression levels changed during rice seed germination. We found that metal transporter genes such as ZIP family has tendency to decrease in their expressions during seed germination. Furthermore, imaging of the distribution of elements (Fe, Mn, Zn, and Cu) was carried out using Synchrotron-based X-ray microfluorescence at the Super Photon ring-8 GeV (SPring-8) facility. The change in the distribution of each element in the seeds following germination was observed by in vivo monitoring. Iron, Mn, Zn, and Cu accumulated in the endosperm and embryos of rice seeds, and their distribution changed during rice seed germination. The change in the patterns of mineral localization during germination was different among the elements observed.     

Proteomic analysis of rice (Oryza sativa) seeds during germination

Although seed germination is a major subject in plant physiological research, there is still a long way to go to elucidate the mechanism of seed germination. Recently, functional genomic strategies have been applied to study the germination of plant seeds. Here, we conducted a proteomic analysis of seed germination in rice (Oryza sativa indica cv. 9311) - a model monocot. Comparison of 2-DE maps showed that there were 148 proteins displayed differently in the germination process of rice seeds. Among the changed proteins, 63 were down-regulated, 69 were up-regulated (including 20 induced proteins). The down-regulated proteins were mainly storage proteins, such as globulin and glutelin, and proteins associated with seed maturation, such as "early embryogenesis protein" and "late embryogenesis abundant protein", and proteins related to desiccation, such as "abscisic acid-induced protein" and "cold-regulated protein". The degradation of storage proteins mainly happened at the late stage of germination phase II (48 h imbibition), while that of seed maturation and desiccation associated proteins occurred at the early stage of phase II (24 h imbibition). In addition to alpha-amylase, the up-regulated proteins were mainly those involved in glycolysis such as UDP-glucose dehydrogenase, fructokinase, phosphoglucomutase, and pyruvate decarboxylase. The results reflected the possible biochemical and physiological processes of germination of rice seeds.