All posts by Chris Wolverton

Fruit Development

  • Fruit development
    • Patterning
      • Carpel development requires AG and SEP genes (MADS box TFs)
      • Genes only in flowering plants, common to all
      • Dry fruits need to have a means of opening, which is
        regulated
    • Carbohydrate import, storage as starch
    • Polyphenolic synthesis
      • Associated with pigment
      • Also associated with lignification of dry fruit carpel wall
  • Fruit ripening
    • Starch conversion to simple sugars
    • Wall breakdown
      • Galactosidases, glucosidases
    • Ethylene signaling
      • Receptor family has five members of two types
        • ETR1 and ERS1 form one type
          • Ethylene binding and dimerization at N terminal
          • His kinase domain at C terminal
            • Like in bacterial two component signaling systems
        • ETR2, ERS2, and EIN4 lack His kinase domain, have
          different kinase domain
      • Signaling pathway
        • CTR1 is a negative regulator
          • Mutant shows constitutive triple response
            Radial swelling, hypocotyl inhibition, hook
            opening
          • Encodes product homologous to MAPKKK proteins

Pollination

  • Pollination
    • self vs non-self recognition
      • Self incompatibility ensures cross-fertilization
      • S locus encodes components of recognition
        • SRK a kinase expressed in stigma PM
          • Found in papillae cells, interacts with SCR
        • SCR expressed in pollen
          • Peptide secreted by microspore and tapetal cells
          • Issues of sporophyte or gametophyte origin
      • Self rejection pathway
        • Pollen complex with SRK endocytosed and degraded
        • Same papilla can detect self simultaneously with cross
    • pollen tube elongation
      • Tip growth
        • Ca++ gradient
        • Actin necessary
      • Water relations
        • Water uptake from surrounding tract
    • egg cell signals

Floral Organ Identity

As long as a shoot apical meristem has a vegetative identity, it continues to produce new leaf primordia that emerge in a particular pattern from the apical dome. When the repression of the floral meristem identity factors AP1 and LFY is released by the induction of flowering, the meristem switches to a program of producing the floral organs: sepals, petals, stamens, and carpels. These organs form in distinct bands, or whorls, that encircle the meristem, which leads to the radial symmetry found in most flowers. Even in flowers showing bilateral symmetry, the same organs are formed in roughly the same whorls, but the organs are often fused in distinctive ways that lead to novel flower shapes. The central question, then, is how does a meristem control the placement of the proper organ in the proper location — how are the floral organs specified?

ABC Model of Floral Organ Formation

Using a genetic approach to answer the question of floral organ identity, researchers have isolated mutants in floral organ formation that show specific defects in the formation of one or more organ. Intriguingly, when an organ went missing, it was often “replaced” by a different organ. For example, in one flowering mutant, the plant failed to form stamens and carpels, and instead replaced them with petals and sepals, respectively. This kind of mutation, where an organ is replaced with another, is known as a homeotic mutation: the replacement of one organ with a different one.

Screening for flower organ mutants eventually uncovered many genes involved in the specification of organ identity, and the genes were often named based on the phenotype of the mutant. The APETALA mutants, AP1, AP2, and AP3, all lacked petals. The PISTILATA (PI) mutant produced excess pistils in place of stamens. Finally, the AGAMOUS (AG) mutant lacked both stamens and carpels, the gamete-bearing organs. As more and more of these mutants were isolated and characterized, a pattern began to emerge that would become known as the ABC model.

 

ABC Model of Flower Development
Diagram of the ABC model of floral organ specification via 3 overlapping domains of influence on meristem tissue identity. Attribution: By Madprime (Own work) [CC0, GFDL (http://www.gnu.org/copyleft/fdl.html) or CC BY-SA 4.0-3.0-2.5-2.0-1.0 (http://creativecommons.org/licenses/by-sa/4.0-3.0-2.5-2.0-1.0)%5D, via Wikimedia Commons
The ABC model predicts that each floral organ is specified by the activity of one or two of the organ identity genes, all of which can be placed into only 3 classes. The ‘A’ class includes AP1 and AP2 activity and specifies the formation of sepals. The ‘B’ class includes AP3 and PI, and specifies the middle two organs (petals and stamens), which we’ll return to in a moment. The ‘C’ class is encoded by AG, and specifies carpel identity.

To explain the homeotic effects, it was postulated that class ‘A’ genes and class ‘C’ genes mutually exclude each other. In cells where the ‘A’ genes are expressed, there can be no ‘C’ gene expression. This phenomenon is common in development, with ‘A’ and ‘C’ known as cadastral genes, setting up a firm boundary between them. Returning to the ‘B’ class, these genes are expressed in an overlapping fashion atop both ‘A’ and ‘C’ regions such that, when ‘B’ mixes with ‘A’, the result is a petal and when ‘B’ mixes with ‘C’, the result is a stamen.

If we return for a moment to the mutant phenotypes, we see that this model explains the main observations of each mutant. For example, the loss of AP1 by mutation results in the pattern carpel-stamen-stamen-carpel. The domain of ‘C’ expression expands into ‘A’ territory, just as would be predicted by loss of ‘A’ activity, resulting in the specification of organs associated with C alone (carpel) or B plus C (stamen). Likewise, agamous mutants show the pattern sepal-petal-petal-sepal, just as predicted by the loss of ‘C’ activity and explained by the encroachment of A activity to take its place. The organs specified are those of A alone (sepal) or A plus B (petal). Finally, in the case of the pistilata mutant, the pattern observed was sepal-sepal-carpel-carpel, just as predicted by the loss of ‘B’ activity.

Since its initial proposal, the ABC model has been enhanced and refined. A fourth class, known as ‘E’, has been added to recognize the contribution of the SEPALLATA genes 1-4. These act as co-regulators in all whorls of the floral meristem, and the loss of all SEP genes results in the formation of only sepals in all organs, thus returning the flower to a collection of leaf-like organs.

The cloning and molecular characterization of the ABC genes revealed them to mostly encode transcription factors. The common thread that ties them together is the nature of the DNA element to which they bind, known as the MADS box. The MADS box is a DNA regulatory region characterized by the sequence CC[A/T]6 GG, and is present in the targets of ABC gene activity. Since their initial discovery in the model plant Arabidopsis, homologs of these MADS-box transcription factors have been identified not only in the flowering plants, but across all eukaryotes.

Transition to Flowering

One of the most significant developmental changes a plant experiences is the switch to reproductive growth from vegetative growth. This transition requires a wholesale change in the identity of the shoot apical meristem, and for most meristems it also means a shift from indeterminate to determinate growth, implying that any new leaf growth must come from another meristem. Given the biological significance of this transition and the horticultural interest in understanding and controlling the timing of flowering, the events that lead to flowering have been intensely studied.

Photoperiod

As with so many other ‘decisions’ made by plants, the inputs that regulate the transition to flowering are made up mostly of cues from the environment. For most plants, the strongest influence over the transition to flowering is day length, and plants can be sorted into 3 classes based on the photoperiod required to induce flowering. Short-day plants transition to flowering when day length begins shortening and reaches a duration less than the critical period for that species. In the northern hemisphere, these are plants that flower after June 21, the longest day of the year. In contrast, long-day photoperiodic plants begin their transition to flowering as day length increases past a certain minimum length. In the northern hemisphere, these are plants that flower in spring and early summer, as day length is waxing. Finally, many plants are day-neutral and will transition to flowering regardless of light cues. Plants that have strict photoperiod requirements for flowering are known as obligate short- or long-day flowering plants, while those that will eventually flower regardless of day length but a particular photoperiod promotes flowering are known as facultative short- or long-day flowering plants.

There is a certain irony in all this talk of day length in promoting flowering, which is that the plant is not actually responsive to the day length at all, but rather to the length of the dark period. In order for obligate short-day flowering plants to begin flowering, they must experience a long, uninterrupted period of darkness. Likewise, obligate long-day flowering plants must experience a shorteningn period of darkness. The reason plants sense the duration of darkness lies in the biophysics of phytochrome, the red/far-red photoreversible photoreceptor. The pool of phytochrome molecules becomes activated by exposure to red light, such as that experienced by a plant during daytime. This pool of molecules slowly reverts back to the inactive red-absorbing form during the dark period, hence, the longer the duration of darkness, the greater the proportion of total phytochrome is inactive. For obligate short-day (or long night) flowering plants, if this dark period is interrupted by even a brief pulse of intense light, that is enough to disrupt the balance of phytochrome signaling that leads to floral transition.

Other cues

A number of other signals besides photoperiod can influence the transition to flowering in some plants. Some plants flower after a given number of days since germination, regardless of the photoperiod. Related to this, some plants transition to flowering after attaining a minimum size — for example, many trees must be 5 or more years old before flowering, probably due to the high resource demands associated with seed and fruit production.

Temperature is another particularly important cue for some plants in the transition to flowering. Many plants require the exposure of the meristem to cold temperatures in order to complete the transition to flowering meristem identity. This process of cold treatment is known as vernalization because of its association with growing through the winter months. The cold treatment has been shown to modulate certain epigenetic factors associated with gene expression, such as DNA methylation. Some plants that have this requirement undergo a two year life cycle with the exposure to cold in the intervening winter. These plants are known as biennial plants, flowering in their second year of growth and typically dying thereafter.

Molecular Signaling

In order to begin making flowers, shoot apical meristems must undergo a change in identity from vegetative to floral (or inflorescence) meristems. This change in meristem identity is influenced by numerous environmental cues, but how do these cues affect the identity of the meristem? Where do the signals come from that result in flower formation, and how do they form?

Through a series of clever experiments starting in the 1920’s, plant biologists have shown that, although the meristem is the site of floral transition, the signals that convert the meristem from leaf-making to flower-making come from the leaves. One of the key experiments to demonstrate this involved grafting a leaf from a plant induced to flower onto a plant that had not yet been induced, resulting in the induction of flowering. For many decades researchers attempted to isolate the diffusible substance causing flowering, but were unsuccessful. Around this same time the plant hormones auxin and gibberellin had been succesfully isolated and characterized, leading many physiologists to search for a similar small organic molecule as the “flowering hormone,” or florigen.

Recent work has revealed that the mRNA and/or protein product of FLOWERING LOCUS T (FT) acts as a positive regulator of flowering. Its expression is upregulated in response to photoperiodic induction in the leaves, and it trafficks through the phloem to the shoot apical meristem. FT expression in leaves is mediated by the expression of another gene, CONSTANS, which encodes a transcription factor that is expressed as an output of the photoperiodic pathway. The movement of FT protein and/or mRNA from the leaves to the meristem requires interaction with a partner called FTIP, or FT interacting protein.

Upon reaching in the meristem, FT interacts with the transcription factor FLOWERING LOCUS D (FD), and together they activate expression of genes that change the identity of the meristem to that of a flower. One example target of the FT-FD dimer is APETALA1 (AP1), a meristem identity gene; another example target is SOC1, which promotes the expression of another meristem identity gene called LEAFY (LFY). Both AP1 and LFY expression are repressed during vegetative growth, and this repression is removed upon arrival of FT in the meristem.

Defense Signaling

One of two major response pathways is initiated when plants are attacked by microbial pathogens. One major difference difference between these two pathways is the specificity with which the plant identifies the pathogen. In one pathway, the plant recognizes general signals common to a broad array of pathogens. These signals are known as pathogen-associated molecular patterns (PAMPs), thus this pathway is called PAMP-triggered immunity (PTI). In the second kind of signaling pathway, the plant responds to highly specific molecules secreted by the pathogen. These molecules are called effectors, hence this pathway is known as effector-triggered immunity (ETI).

PAMP-triggered immunity (PTI)

The hallmark of PTI is the ability of the plant to detect the molecular signature of pathogens. These signatures may take many different forms, including peptides derived from bacterial flagellum proteins, chitin (which makes up the cell wall of fungi), double-stranded RNA, and certain sequences of DNA common to microbes. Each of these presents a specific molecular pattern recognizable by the plant as indicating the presence of a potentially pathogenic organism.

These molecular signatures are detected by the cell through a class of proteins known as pattern recognition receptors (PRRs) that are specific for certain molecules. For example, the flagellum peptide known as flg22 is perceived by a PRR called FLS2. It is important to note that the flg22 peptide is a common motif found in all bacterial flagella as they are hydrolyzed. Upon perceiving flg22, FLS2 interacts with and phosphorylates the first member in a kinase cascade called MEKK1. MEKK1 phosphorylates one or more of several kinases (MEK4/5), which target several other kinases (MPK3/6). This series of events is known as a mitogen-activated protein kinase cascade, a signal transduction pathway common across all organisms. This kind of signaling system is modular and achieves the amplification of a single signal. The end result of all of this kinase activity is the activation of several transcription factors (WRKY22/29) that control defense-regulated genes. One example of these genes is those involved in synthesis of salicylic acid and jasmonic acid. There is also evidence that the kinase cascade can modify chromatin structure through acetylation and methylation, both of which can have a strong effect on genome-wide gene expression. All of this signaling has the overall result of “priming” the plant defense response.

Effector-triggered immunity (ETI)

The fundamental difference between PTI and ETI is the degree of specificity, with ETI representing a highly-specific, gene-for-gene defense response. The high specificity makes this pathway less durable than PTI and more targeted against an individual pathogen.

The ETI response is initiated by the secretion of effector molecules by the secretory system of bacterial cells. These effector molecules, called Avr (avirulence) proteins, are detected by the plant through recognitional resistance genes, or R-genes. The R genes encode Ser/Thr protein kinases, which interact with partners called nucleotide binding leucine-rich repeat proteins (NB-LRRs). This signaling complex also touches of a kinase cascade, but the overall result is the activation of the hypersensitive response and rapid cell death around the site of infection.

The avirulence (Avr) proteins represent an interesting evolutionary riddle: how is it adaptive for the bacteria to secrete a molecule that signals its presence to the plant? In at least one case, the answer seems to be related to the function of the Avr protein. AvrAC, secreted by a strain of Xanthomonas, acts as a uridyl transferase that modifies a component of the signal cascade such that its phosphorylation site is masked. Unable to be phosphorylated, the kinase cascade is interrupted and the plant fails to activate the hypersensitive response.