Sequentially happened activities during rice seed germination.
Upon imbibition, rice seed germination process could be divided into three phases (Yang et al., 2007). Phase I is the first rapid water-uptake period with the onset of mRNA biosynthesis (Howell et al., 2009), phase II is the most important stage for metabolism reactivation, mobilization of reserves, cell structure repair, cell wall loosening, and coleoptile elongation, phase III is another rapid water-uptake stage with TCA and aerobic respiration recovering, cell division initiation, radical protrusion, and initiation of seedling establishment. The picture displayed the rice seed after 0, 24, 48, and 72 h imbibition (provided by Mr. Chao Han).
During seed germination, the increasing of total water content or fresh weight follows a classic triphasic model (Bewley, 1997). When germinating in the distilled water, the rice seed weights increased rapidly during the first 20 h imbibition (phase I), and there is no significant morphology changes. The phase I is followed by a stable plateau stage until 50 h (phase II) during which the coleoptiles elongation could be observed at this stage. Phase III is another rapid water uptake stage accompanying with the protrusion of the radical (Yang et al., 2007; Figure ). Phase II was usually regarded as the most important stage, because all of the germination required metabolic reactions are reactivated during this period. However, transcriptome of germinating rice seed indicated that the switch may happen even earlier, since a greater proportion of immediate transcript has been identified at this point (Howell et al., 2009).
Proteins Involved in Rice Seed Germination
Upon imbibition, vivid rice seeds should reboot the system activity and mobilize the reserves for germination. Since protein is the real executor of life activities, a series of proteins will participate in the germination process. Some indispensable proteins for germination will be discussed here, such as those related to metabolic, storage, protein synthesis, ROS scavenger, signaling to name a lot.
Without exception, metabolism-related proteins, especially those involved in the carbohydrate metabolic pathways including major and minor carbohydrates metabolism, glycolysis, TCA cycle, fermentation, gluconeogenesis and glyoxylate cycle and pentose phosphate pathway (PPP), were the major group of proteins existing in germinating rice seed (Figure 3). Since starch is the major reserve of the rice endosperm, all the enzymes involved in starch degradation to hexose phosphate were identified in rice seeds at 24 h of germination. Except for α- amylase that was greatly increased in abundance at the late stage of phase II, all the other enzymes keep constant in the whole germination process (Yang et al., 2007; He et al., 2011a), which suggested that the starch degradation metabolism is vigorous at the phase II. The hexose phosphate from the degradation of starch will further experience glycolysis. Totally, 22 enzymes that catalyze all the steps in the glycolysis pathway were detected, and most of them were up-regulated at 12 h imbibition. Pyruvate, the final product of glycolysis, could be further degraded through TCA cycle; most TCA cycle-related proteins were also identified as up-regulated proteins (Yang et al., 2007). Kim et al. (2009) had detected that both succinyl-CoA ligase and cytoplasmic malate dehydrogenase were stably accumulated during germination. TCA cycle might, along with glycolysis, provide the main energy at the late stage of germination. Due to lack of functional mitochondria, the aerobic respiration was detected very low during the first 48 h of imbibition. Anaerobic respiration pathway, such as fermentation, might be the main source of energy at the early stage of germination, which is supported by the identification of lactate dehydrogenase (LDH), pyruvate decarboxylase and alcohol dehydrogenase during this period (He et al., 2011a,b;Yang et al., 2007). Most of the enzymes involved in the PPP also existed in the germinating seeds, and the glycolytic enzymes were identified to be carbonylated and degraded during germination (Job et al., 2005). Blocking glycolysis and reorienting the glucose flux to the PPP could provide cells not only with reducing power in the form of NADPH to surmount oxidative stress, but also pentose phosphate for nucleotide metabolism (Arc et al., 2011).
To be mentioned, all the enzymes related to starch biosynthesis also exist in the embryo of germinating rice seeds. The accumulation of starch granules had been observed in rice embryos during germination, which suggested the solute from the endosperm can be re-synthesized to starch at the embryo (Matsukura et al., 2000;He et al., 2011a,b). But how those solutes were transferred from endosperm to embryo is not understood well. To reboot the quiescent system, de novo synthesis of new functional proteins is necessary. Enzymes that related to protein biosynthesis, modification, targeting, and folding, including ribosomal proteins, translation initiation or elongation factors, amino acid activation proteins, trafficking, and secretion proteins and chaperonins, were identified during germination. On the contrary, the storage proteins, seed maturation associated proteins, embryogenesis proteins and proteins related to desiccation were degraded to provide primary amino acids and reduced nitrogen for the seed germination. Although the lipids are not the major reserves, the storage lipids did accumulate during the grain filling and stored in aleurone cells of the cereal seeds (Krishnan and Dayanandan, 2003). β-oxidation and glyoxylate cycle are the two major fatty acids degradation pathways. Enzymes involved in these two pathways were detected during rice seed germination. Interestingly, fatty acid biosynthesis related enzymes such as ATP: citrate lyase and some transacylase had also been detected. Most of amino acids biosynthesis and degradation related proteins also had been identified during rice seed germination. Since most of the biosynthesis and degradation metabolisms are in concurrence, we may wonder when storage reserves should be mobilized during seed germination. Gallardo et al. (2001) inferred that the potential mobilization might exist not only in germination but also in the maturation phase based on the fact that both precursor forms and proteolyzed forms of the 12S seed storage-protein subunit were identified in dry mature seeds.
Reactive oxygen species (ROS) are produced in all living organisms. The ROS might be bifunctional, and over accumulation of ROS can result in oxidative stress. Reducing the oxidized proteins is another critical way to cope with ROS. Upon imbibition, the contents of ROS were gradually increased. ROS can be efficiently scavenged by the antioxidant enzymes like superoxide dismutases (SODs), glutathione S-transferase (GST), catalase, peroxidases, and enzymes in the ascorbate–glutathione cycle. Many of these redox regulation proteins were identified during germination (He et al., 2011a). Carbonylation, one of the important post-translational modifications (PTMs) under oxidative stress (Nystrom, 2005), can not only provoke the degradation of reserve proteins, but also be fatal by targeting those physiological important proteins (Job et al., 2005). Meanwhile, accumulation of NO upon seed imbibition can help to regulate the redox homeostasis by S-nitrosylation of the critical protein thiols and protect them from oxidation (Lounifi et al., 2013). So keeping the redox homeostasis is necessary in vivo.
Stress responsive proteins were accumulated during the seed maturation in order to survive from dessication. Among them, HSPs were the largest group. Proteins including universal stress proteins (USP), dehydrins, DNA J family, and late embryogenesis abundant (LEA) proteins can also be regarded as desiccation stress responsive proteins (Yang et al., 2007; He et al., 2011b). The metabolism of nucleotides was not active at early stage of germination since few enzymes required for nucleotide metabolism were detected. However, all 8 except proteins for polygalacturonase proteins involved in the cell wall biosynthesis were identified at 24 h after imbibition, which implied cell wall biosynthesis is active during the phase II (He et al., 2011b; Sano et al., 2012).
The seeds are carried away from the mother plant by animals called dispersers. In some cases the animal carries away an entire fruit, in other cases the animal is carrying seeds alone. The animal usually feeds on the fruit (ovary wall), but “tosses” the seeds (or passes them through its digestive system). This assures that seeds end up far away from the “mother” plant, where they can develop without competition from “mom.”
Having been taken away from the parent, it is time for the seeds to sprout or germinate. Seed germination is defined as the emergence of the radicle through the seed coat. Common garden seeds germinate if given just water and reasonably warm temperatures.
Wild species usually have some kind of deeper dormancy to avoid sprouting in late summer or fall when the seeds are commonly dispersed. This assures that tender seedlings are not frozen at a young age by the approaching winter. Instead the seedlings do not appear until warm weather arrives in springtime.
If a species has evolved a very thick seed coat, it may require scarification of that seed coat before water can enter the seed and initiate germination. Perhaps the coat has to be gnawed at by animals, or frozen and thawed repeatedly to crack the coat, or rund down the rocks in a stream bed or up beach in pounding surf to wear the coat down. Perhaps the seed is swallowed whole by the disperser. The thick seed coat protects the dormant embryo as it passes through the animal’s digestive system. The weakening of the seed coat through digestion means that the seed is ready to sprout when it gets deposited (along with a bit of fertilizer) by the disperser somewhere new in the environment. Yet other seeds require a brief exposure to fire to scarify the seed coat.
In species with thin seed coats, light may be able to penetrate into the dormant embryo. The embryo may then either use the presence of light or the absence of light to trigger its germination process. Small seeds with thin seed coats are likely to use light as a signal for germination. If these are buried shallowly enough for light to hit the seed, then its germination will initiate and before it runs out of reserves, its cotyledons will be doing photosynthesis. If the small seed is deeply buried it does not germinate; it has no idea how deep it is buried and may not have sufficient reserves on board to make it to the surface before they run out. Evolution favors shutting down germination in the dark for those species. For large-seeded species, the opposite may be true. There is an advantage to having your reserves buried before starting to germinate…otherwise the sprout just acts as a flag to say…”come and eat my nutritious seeds here on the surface.” Evolution favors a large seed with lots of investment in storage to wait until it is buried before sending up its shoot. Such a seed will key in on darkness rather than light. In laboratory you will see an example of each of these kinds of evolutionary adaptations!
In yet other species, the embryo is fully developed when the seeds are dispersed. They need to be deposited in soil that possesses fungi that serve as host to the seed and seedling until the development is completed and the seedling is big enough to grow on its own. Trying to sprout many orchid seeds in common garden soil is futile…the required fungal hosts are not present…so leaving wild orchids where they naturally grow is the best idea.
In most species of wild plants the mother invests the seed with abscisic acid. This hormone makes the embryo dormant until environmental conditions and native enzymes permit the abscisic acid to be broken down inside the embryo. The enzyme required is present in the embryo in the fall, but it is inactive until activated by low temperature. About four weeks at 4°C is required to activate the enzyme and degrade all the abscisic acid in the embryo. This cold treatment is often called stratification. Then the seed is ready to germinate, but it is too cold to do so. Vernalization or bringing the cold-treated seed into warm spring-like conditions will then allow the germination process to begin. This way seeds that have evolved in areas with cold winters avoid germinating until spring!
For desert plants, the role of abscisic acid it taken instead by phenolic compounds. These chemicals inhibit seed germination too, but they are not broken down by cold weather. Instead phenolics are water soluble and need to be leached out of the seeds by repeated washings to initiate germination. For a desert plant, this mechanism has evolved to ensure that seeds do not begin to germinate until the wet season has certainly arrived. A single passing shower in the dry season will not do…it takes several soaking rains to do it.
Examples of various mechanisms are summarized in the figure below.
Also shown above is a mechanism for seed germination in two species: Barley and lettuce. That is a bit misleading as the sketch clearly shows barley (a monocot) and not lettuce (a dicot) so read carefully below:
The barley seed shown is a typical monocot. It has a seed coat (fused to fruit coat), a large endosperm area filled with starch, and an embryo (or germ). Barley lacks any special dormancy…germination is initiated by water and reasonably warm temperature. The seed takes up water from the environment in the process known as imbibition. The water passes through the embryo, picking up the germination signal: the hormone Gibberellic Acid. The water moves the hormone from the embryo to the aleurone layer of the endosperm. This layer of cells stores much protein and is the “brown” of “brown rice.” When cooked, this protein-rich layer gives brown rice its chewiness. The water activates hydrolysis enzymes that degrade the storage protein into amino acids. The gibberellic acid activates the DNA gene coding for the enzyme amylase in the aleurone cells. Transcription in the nucleus and translation by ribosomes in the cytosol results in the production of amylase inside the aleurone cells. The amino acids from hydrolysis of storage protein are used in the translation of amylase. The amylase is shipped by ER into the Golgi, sorted and packaged into vesicles, and exported through the cell membrane by exocytosis. The amylase is thus dumped into the endosperm interior. There the amylase catalyzes the hydrolysis of starch into sugar. The sugar happens to be maltose, which is transported to the embryo. The sugar fuels respiration in the embryo so it can grow. The radicle protrudes from the seed coat, and germination is accomplished in barley.
A similar mechanism exists in lettuce. The picture above is only symbolic. Lettuce is a dicot, it has no aleurone layer, and the starch is mostly found in the embryo rather than in endosperm. Rather than gibberellic acid, the activating signal is a pigment called phytochrome. This pigment exists in two different chemical forms: Pr and Pfr. How a lettuce seed responds depends on how much of each of these two forms is present in each cell. The active form of phytochrome is Pfr. If there is sufficient Pfr in the seed when the imbition takes place, it will photoactivate the genes for amylase and the seed will sprout using the mechanisms shown in the diagram above.
Typical lettuce seed batches germinate between 5% and 20% if placed in darkness because at least this many seeds have enough Pfr to stimulate germination without any help from humants. If, however, you put the lettuce seeds in red light (660 nm), the red light causes all the Pr to change into Pfr. Now 85-95% of the seeds can sprout because they all have an abundance of Pfr inside. On the other hand, if you put lettuce seeds in far-red (730 nm) light, the far-red light causes all the Pfr to change into Pr. In far-red light, then, all the seeds have essentially no Pfr and so very few (0-5%) actually sprout. You carried out these experiments in lab, or soon will.