SEED & FRUIT DEVELOPMENT
 
SENESCENCE - An introduction to senescence involves the terms monocarpic and polycarpic behavior.  Some plants die at the time when their fruits mature i.e. cereals, peas, soybeans, etc. and this is called monocarpic senescence.  This behavior is distinguished from polycarpic species which go through many cycles of growth and reproduction before death.  Cereals are characterized by having all vegetative apices transformed into determinate reproductive structures.  Others like soybean show a close correlation between seed-filling and senescence and this lead to the Nutrient Diversion Hypothesis.  This hypothesis is based largely on the observation that removal of flowers or developing fruits will delay senescence.  There are many exceptions to this hypothesis and it is an over-simplification, but useful in thinking about mechanisms.  ABA applied to leaves of fruiting soybean plants accelerated their senescence, but did not enhance the senescence of depodded plants.  Thus ABA does not seem to be the senescence-inducing factor.  It is in fact carried preferentially from leaves to fruits.  The role of hormones in regulating senescence usually involves two aspects: (1) elimination of sources of auxin as vegetative shoot apices are converted to flower development and young leaves mature; and (2) a hypothetical factor for senescence (SF) which is produced by developing fruits and promotes senescence.  In addition, cytokinins perpetuate the vegetative condition and also decline with auxin.  Developing fruits not only produce SF, but also ethylene.

ETHYLENE - Review the ethylene biosynthetic pathway and recall that the two important enzymes are ACC synthase and ACC oxidase.  Also that ACC synthase is encoded by a multi-gene family, and certain members are expressed during fruit ripening.  Ethylene is involved not only in senescence, but also in germination, cell elongation, fruit ripening, and other aspects of growth and development.  In Arabidopsis ethylene sensing involves a protein kinase transduction pathway.  This pathway has two genes, one encodes a protein with similarity to the Raf family of Ser/Thr protein kinases, the other most likely encodes an ethylene receptor that functions in a pathway similar to the prokaryotic two-component histidine kinase system.  Recall that the triple response of seedlings grown in the dark is an ethylene response, and mutants that fail to display this response, even in the presence of exogenous ethylene, are likely to be unable to produce the ethylene receptor.  These ethylene insensitive mutants appear as tall seedling above a lawn of short wild-type ones.  The ETR1 gene encodes a membrane-associated disulfide-linked dimer protein that is suggested as the receptor for ethylene.  Review the other mutants which are defective in the regulation of ethylene biosynthesis, or at various places in the transduction pathways leading to the many ethylene responses.  A number of auxin resistant mutants also display resistance to ethylene.

SEED DEVELOPMENT - A generalized representation of embryo development in angiosperms from zygote to seed usually includes: embryogenesis; maturation; and desiccation.  Embryo development is accompanied by reserve food accumulation, and the synthesis of seed storage proteins is the most intensively studied.  The maturation loop can be bypassed in culture by precocious germination of embryos.  This occurs in the absence of ABA and the presence of reduced nitrogen and carbon (i.e. glutamine & sucrose).  In the presence of high osmoticum and/or ABA , maturation is promoted and precocious germination inhibited.  Upon desiccation, which often involves a water content change of 85% to 10%, these processes are finalized, and normal germination will occur in response to imbibition.  In culture, embryos can reversible enter or leave the maturation loop by application or removal of high osmoticum or ABA.  The role of various hormones during embryogenesis has been studied and can be summarized as follows: early stages are promoted by low concentrations of auxin, inhibited by high concentrations, CKs enhance the cell cycle especially in endosperm tissue, both GAs and CKs play a role in suspensor development and supply hormones to the embryo; mid-stages show high levels of both IAA and GAs during cell expansion and fruit growth, mutants suggest that low levels of GAs are more effective, ethylene has a role in seed germination rather than during seed development; late embryogenesis shows high levels of ABA and decreasing levels of GA and IAA as desiccation occurs, ABA decreases in dry seed and inhibits germination, ABA-insensitive mutants exhibit vivipary and fail to develop ABA-inducible proteins, the role of ABA in development is not clear and it is thought that it plays no role in dicot maturation program.  Review the ABI-3, LEC1, and FUS3 genes in Arabidopsis and Fig. 11.10 for a summary of the genetic regulation of events in late embryogenesis based on these mutants.

Seed Storage Proteins - seed storage proteins are synthesized in large amounts and occur in protein bodies in cotyledon or endosperm cells.  Those found in aleurone cells are different and contribute little to storage proteins.  Each plant produces a characteristic array of storage proteins, which are classified on such characteristics as size, solubility, and amino acid composition.  They are encoded by small gene families and mutations have major effects on their amounts and amino acid composition.  The mutation opaque-2 in maize seems to be regulatory and controls a cluster of structural genes.  An important paper is that of N. Murai et al.,1983, given in the readings for this week, since it was the first example of a specific gene transferred from one plant to another via a vector system..The gene encoded phaseolin, a glycoprotein constituting up to 50% of the storage proteins in bean.  Since this paper a number of seed storage protein genes have been cloned and used to make transgenic plants.

SEED GERMINATION - review the general aspects of seed germination including the imbibition of water, cell expansion, hydrolysis and transport of food reserves in endosperm or cotyledons. The food reserves may include starch, fat, protein, or some combination, plus other compounds like phytin, which contains phytic acid, a hexaphosphate of inositol where the ring hydroxyls have become esters of phosphoric acid, then K+, Ca2+, or Mg2+ salts.  There are many different types of dormancy including immature embryos and those requiring an after-ripening period, various characteristics of the seed coat like hard and impermeable, a variety of light effects, and a variety of temperature requirements.  Review the viviparous mutants of Arabidopsis and their role in dormancy and the control of germination.   Seeds that store lipids usually require the biogenesis of the glyoxysome for gluconeogenesis.  Recall the famous Rapp & Randall scheme for the subcellular and metabolic reactions of fatty acid breakdown and the synthesis of sucrose and new fatty acids in castor bean endosperm.  The complex interplay between  glyoxysomes, mitochondria, proplastids, and the cytoplasm, as well as key enzymes like isocitric lyase (de novo in many species), malate synthase, and others, make this an interesting and complex story.  This would also be a good time to review the GA/alpha amylase etc. story in barley aleurone cells.

FRUIT RIPENING - Fruit ripening involves the events that lead to changes in the color, softness, aroma, and flavor in various fruits.  Some common features of these processes include  the biosynthesis of ethylene and especially the concept of two systems for ethylene production.  Fruits can be classified according to whether they are climacteric or nonclimacteric.    System I ethylene biosynthesis is common to climacteric and non-climacteric fruits, and System II ethylene biosynthesis is found only in climacteric fruits and is termed autocatalytic since after exposure to ethylene there is a stimulation of further ethylene production.  This induction results in massive increases in ethylene production.  Ethylene is the central factor in fruit ripening, ripening fruit produce ethylene and are in turn induced to ripen by ethylene.  Thus to delay or inhibit ripening a block in ethylene production in vivo should work.  Various antisense genes that inhibit synthesis of ethylene were studied and transformed tomato plants with a antisense ACC oxidase gene synthesized far less ethylene that wild type and fruit ripened very slowly.  Review the paper of Oeller, et al.  1991, found in the reading list, which gives the details of how transformed tomato plants with an antisense ACC synthase gene specific to an ACC synthase enzyme that is expressed in ripening fruit was shown to suppress ethylene synthesis, some 99.5%. Such transgenic fruit remained unripe until treated with exogenous ethylene, when they ripened into normal fruit.  Thus ethylene appears to be the inducer for ripening and not just some by-product of ripening.  Other constructs that had various controls on ethylene biosynthesis also were shown to work, including an ACC deaminase from soil bacteria.  These transgenic plants also allowed the analysis of other genes and their products for their role in ripening.  One example is polygalacturonase, a key enzyme in fruit softening, which had normal expression in the antisense ACC synthase transgenic plant, and thus may not be solely responsible for the softening response in ripening.

Fruits develop from ovaries and this development is triggered by pollination or fertilization.  In fleshy fruits, such as tomato, the bulk of the fruit is derived from L3.  Only the epidermal and subepidermal layers are derived from L1 and L2.  A good  review of this process is found in Gillaspy, J. et al.  1993.  Fruits: a developmental perspective.  Plant Cell 5:1439-1451.  The development and ripening of commercial fruit have been staged for growers, processors, and sellers, and has been divided into three phases.  Phase I occurs at anthesis and involves ovary development, fertilization, and fruit set.  Cell Cycles occur in Phase II when seeds form and embryogenesis occurs.  In Phase III, cells of the fruit expand and the embryos mature.  The onset of cell expansion of tomato fruit growth coincides with a peak in auxin accumulation.

ABA - This seems like a good time to review ABA signal transduction in seeds, since this was the subject of a recent review in the ARPPPMB, the citation is ABA Signal Transduction by J. Leung & J. Giraudat, 1998, 49:199-222.  Endogenous ABA content peaks during the last 2/3 of seed development, then lowers in the dry seed.  ABA has been shown to regulate the accumulation of food reserves,  the acquisition of desiccation tolerance, and the induction of seed dormancy.  It is known that exogenous ABA inhibits precocious germination of immature embryos in culture, and that in some cases of vivipary the seeds are not able to synthesize normal amounts of ABA, and that both cases are related to ABA biosynthetic mutants.  Thus it is clear that endogenous ABA inhibits precocious germination and promotes seed dormancy.   Genetic analysis of signaling elements that mediate regulation of seed dormancy and germination has revealed a variety of mutations that alter sensitivity to ABA in both maize and  Arabidopsis.    Several of these genes have been cloned and studied.  Some vivipary embryos do not have reduced ABA content, but rather a reduced sensitivity to germination inhibition by exogenous ABA in culture.  One such gene is VP1 of maize which encodes a seed-specific transcription factor.  The ABA-insensitive mutants of   Arabidopsis   ABI1, ABI2, etc. were identified by selecting seeds able to germinate in the presence of 3-10 micromolar ABA, a concentration which is inhibitory to wild type seeds.  These mutants also exhibit a marked reduction in seed dormancy.

Other mutants termed ERA or Enhanced Response to ABA types were identified by lack of germination in the presence of low concentrations of ABA (0.3 micromolar) that are not inhibitory to wild type germination.

The accumulation of reserve foods and the acquisition of desiccation tolerance have also been subjected to mutational analysis.  The expression of specific sets of mRNA encoding storage proteins and late-embryogenesis-abundant (LEA) proteins encoding factors involved in the acquisition of desiccation tolerance can be induced by exogenous ABA in cultured embryos.  ABA-deficient mutants also support similar activities.  Two classes of transcriptional elements have been identified: one class is Cis-acting promoter elements involved in the activation of gene expression by ABA; another class includes Trans-acting factors and upstream signaling elements.  Thus there is beginning an accumulation of  genetic evidence that may allow the elucidation of the transduction pathway(s) for ABA in the control of the accumulation of food reserves, the acquisition of desiccation tolerance, and the induction of seed dormancy.  Recall also that ABA will reverse the GA  induction of an increase in free calcium ions and alpha-amylase in barley aleurone prototplasts.  A process which initiates the germination response in barley and other grains.

Signal transduction in animal systems for epidermal growth factor (EGF) follow this simplified scheme: Receptor Kinase -> Ras-> Raf->MEK-> MAPK (mitogen-activated protein kinase).  Plants, like animals, also balance the processes of the cell cycle and cell differentiation, and they show many parallels to signal transduction in animals.  Ethylene induces the sequential phosphorylation of several proteins that have counterparts in animal cells.  The ethylene receptor cannot be directly linked to animal cell receptor kinases like EGF receptors, however, other of the cellular responses to ethylene seem to depend on a number of orthologs of animal signal transducing proteins.  Kinases of the Raf, MEK, and MAPK types have all been directly implicated in ethylene signaling, although as negative regulators.