Regulation of Floral Development (Part III)

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PHYTOCHROME AND PHOTOPERIODISM

Phytochrome

•Phytochrome is a protein pigment that absorbs red and far-red light most strongly, but that also absorbs very low amount of blue light.

Phytochrome can interconvert between Pr and Pfr forms:

•Phytochrome is synthesized in a red light–absorbing form (Pr) which is blue to the human eye.

•Pr is converted by red light to a far-red light–absorbing form (Pfr) which is blue-green.

•Pfr, in turn, can be converted back to Pr by far-red light.

•This phenomenon is known as photoreversibility.

•Darkness also converts Pfr to Pr.

•Pfr is the physiologically active form of phytochrome.

Photostationary state

•The proportion of phytochrome in the Pfr form after saturating irradiation by red light is only about 85%.

    Reason: Most of Pr absorb red light and are converted to Pfr, but some Pfr also absorbs red light and are converted back to Pr

•Similarly, under far-red light irradiation, an equilibrium of 97% Pr and 3% Pfr is achieved.

    Reason: Very small amount of far-red light absorbed by Pr makes it impossible to convert Pfr entirely to Pr by far-red light.

•This equilibrium is called a photostationary state.

Phytochrome is a dimer composed of two polypeptides

•Native phytochrome occurs as a homo-dimer.

•Each subunit consists of:

        •a light-absorbing pigment molecule called the chromophore

        •a polypeptide chain called the apoprotein.

•The chromophore is attached to the apoprotein through a thioether linkage to a cysteine residue

•Both the chromophore and protein undergo conformational changes during Pr-Pfr interconversion.

PHY genes encode two types of phytochrome:

•Phytochrome is encoded by a multigene family consisting of five members: PHYA, PHYB, PHYC, PHYD, and PHYE.

Type I phytochrome:

•Encoded by PHYA gene

•Transcriptionally active in dark-grown seedlings

•Expression is strongly inhibited in light in monocots and less dramatically in dicots.

•PfrA is also unstable.

Type II phytochrome:

•Encoded by PHYB, PHYC, PHYD, PHYE genes

•Expression of their mRNAs is not significantly changed by light.

•Proteins are more stable in the Pfr form.

Phytochrome is the primary photoreceptor in photoperiodism

In SDPs, a night break becomes effective only when the supplied dose of light (Red light) is sufficient to saturate the photoconversion of Pr to Pfr.

•A subsequent exposure to far-red light, which photoconverts the pigment back to the physiologically inactive Pr form, restores the flowering response.

Reason:

      •During night, Pfr gets converted to Pr.

    •A long night length is required to convert sufficient amount of Pfr to Pr.

   •If a flash of red light is given, Pr absorbs red light and gets converted back to Pfr.

    •So, the effective night length gets reduced and sufficient Pfr is not generated.

    •If the flash of red light is followed by a flash of far-red light, the Pfr again gets converted to Pr and this nullifies the effect of red light flash.

                      Pfr/Pr > 1   …….Flowering in LDPs

                      Pfr/Pr < 1   …….Flowering in SDPs

•In LDPs, a night break of red light promoted flowering, and a subsequent exposure to far-red light prevented this response.

A Blue-Light Photoreceptor Also Regulates Flowering

•In some LDPs, such as Arabidopsis, blue light can promote flowering.

•Cryptochrome is the major blue light photoreceptor that regulates flowering.

GENETIC AND MOLECULAR REGULATION OF FLOWERING

Transition to flowering involves multiple factors and pathways

Five genetically distinct developmental pathways control flowering. These are:

        ØPhotoperiodic pathway

        ØVernalization pathway

        ØAutonomous pathway

        ØSucrose pathway

        ØGibberellin pathway

Photoperiodic pathway

It involves phytochromes (PHYA and PHYB acting antagonistically) and cryptochromes.

In Arabidopsis (LDP)

•In the morning, PHYB represses CONSTANS (CO) gene expression.

•In the evening, PHYA and cryptochrome interacts with circadian clock genes to induce CO expression.

•CO is a transcription factor which induces FLOWERING LOCUS T (FT).

•FT protein (‘Florigen’) travels from the induced leaf to the shoot apex via the phloem.

•FT forms a complex with transcription factor FD.

•FT-FD complex activates downstream genes like SOC1, LFY, etc which in turn activates the floral homeotic genes for floral development.

In rice (SDP)

•In the morning, PHYB represses Heading date 1 (Hd1) which is the homologue of CO.

•In the evening, PHYA interacts with circadian clock genes to induce Hd1 expression.

•Here, Hd1 acts as an inhibitor of flowering.

•Absence of Hd1 stimulates expression of Hd3a.

•Hd3a (‘Florigen’) travels from the induced leaf to the shoot apex via phloem for floral development.

Coincidence model

•Transition to flowering occurs when the expression peak of CO coincides with the light phase.

•PHYA/PHYB perceive light signals and entrain the clock

•The clock genes control the diurnal expression of CO and its expression peaks between 12 hours of light and dawn (SuaÂrez-LoÂpez et al., 2001; Nature).

•In long days, the CO expression peak coincides with the light phase (coincidence phase). 

•In light, the CO protein is stable and can activate FT expression, promoting flowering. 

•In short days, CO expression peaks in the dark (no coincidence of light phase and CO expression). 

•In the dark, CO protein is degraded and thus cannot induce FT expression and further processes.

(Singh et al., 2016; New Phytologists)

Florigen concept

•During flowering, a biochemical signal is produced in photoperiodically induced leaves and is then transmitted to the shoot apex where it stimulates flowering.

•This mode of action resembles a hormonal effect.

•Hence, Mikhail Chailakhyan postulated the existence of a universal flowering hormone, florigen (1930s).

•Evidence: non-induced receptor plants were stimulated to flower by being grafted onto a leaf or shoot from photoperiodically induced donor plants.

•Movement of the floral stimulus from a donor leaf to the stock across the graft union correlated closely with the translocation of 14C-labeled assimilates via vascular continuity across the graft union.

Identification of florigen

•Since 1930s, many attempts to isolate florigen have remained unsuccessful.

•A major breakthrough is the identification of FLOWERING LOCUS T (FT) in Arabidopsis.

•FT is a downstream target of CO and is expressed in companion cells of leaves (like CO).

•FT is a small globular protein induced by CO in response to long days.

•FT then travels from leaf to shoot apex via phloem to induce flowering.

•It can travel through grafting unions and induce flowering in competent recipient plants.

•Therefore, FT (or its homologue, Hd3a in rice) is the florigen.

Regulation of floral induction by FT

•FT mRNA is synthesized in leaf companion cells.

•Endoplasmic reticulum protein, FT INTERACTING PROTEIN 1 (FTIP1), helps the transport of FT  from companion cells to sieve tubes.

•FT protein then moves via phloem translocation stream to shoot apex.

•In the shoot apex (floral meristem), FT enters the nucleus to bind FLOWERING LOCUS D (FD) which is a transcription factor.

•FT-FD complex activates floral identity genes like SOC1 and AP1.

•SOC1 then activates LFY which further activates the floral homeotic genes for floral development.

VERNALIZATION: PROMOTING FLOWERING WITH COLD

•Vernalization is the process whereby flowering is promoted by a cold treatment given to a fully hydrated seed (i.e., a seed that has imbibed water) or to a growing plant.

•Commonly, a combination of cold treatment followed by long-day exposure is required.

Vernalization results in the competence to flower at the shoot apical meristem

•The effective temperature range for vernalization is from just below freezing to about 10°C.

•The effect of cold increases with the duration of the cold treatment until the response is saturated.

•Vernalization can be lost as a result of exposure to devernalizing conditions, such as high temperature, but the longer the exposure to low temperature, the more permanent the vernalization effect.

•Vernalization takes place primarily in the shoot apical meristem.

•Localized cooling causes flowering when only the stem apex is chilled and is largely independent of the temperature experienced by the rest of the plant.

•In case of seed vernalization, fragments of embryos, consisting essentially of the shoot tip, are sensitive to low temperature.

•Vernalization results in the acquisition of competence of the meristem to undergo the floral transition.

Vernalization involves epigenetic changes in gene expression

Changes in gene expression that is stable even after the signal that induced the change (in this case cold) is removed are known as epigenetic regulation.

•The involvement of epigenetic regulation in the vernalization process has been confirmed in the LDP Arabidopsis.

•In Arabidopsis, a gene, FLOWERING LOCUS C (FLC), acts as a repressor of flowering.

•FLC is highly expressed in non-vernalized shoot apical meristems and negatively regulates SOC1.

•After vernalization, FLC is epigenetically (conversion from euchromatin to heterochromatin by histone methylation) switched off permitting flowering in response to long days to occur.

•In the next generation, however, the gene is switched on again, restoring the requirement for cold.

Autonomous pathway

•Here, flowering occurs in response to internal signals—the production of a fixed number of leaves, age, etc.

•The autonomous pathway acts by reducing the expression of the flowering repressor gene FLOWERING LOCUS C (FLC), an inhibitor of LFY.

Sucrose pathway

•Sucrose stimulates flowering in Arabidopsis by increasing LFY expression.

Gibberellin pathway

•Required for early flowering and for flowering under non-inductive short days.

•GA induces GA-MYB (transcription factor) mediated activation of LFY.

 

(Images kindly shared by Oxford University Press and are being used for teaching purpose only)

Regulation of Floral Development (Part II)

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FLORAL EVOCATION: INTERNAL AND EXTERNAL CUES

Flowering is regulated by:

ØInternal factors controlling the switch to reproductive development:

•Age

•Size

•Number of leaves

Ø External cues controlling seasonal response:

•Photoperiodism (Light/dark cycle) and  light quality (wavelength and intensity)

•Vernalization (Low temperature)

•Total light radiation

•Water availability

•External cues and internal developmental factors together fine-tune flower evocation in most plants.

•When flowering occurs strictly in response to internal developmental factors and does not depend on any environmental conditions, it is referred to as autonomous regulation.

THE SHOOT APEX AND PHASE CHANGES

•In higher plants, developmental changes occur in a single, dynamic region, the shoot apical meristem.

•During postembryonic development, the shoot apical meristem passes through three well-defined developmental stages:

ØThe juvenile phase

ØThe adult vegetative phase

ØThe adult reproductive phase

•The transition from one phase to another is called a phase change.

•Juvenile tissues are produced first and are located at the base of the shoot.

Competence and determination are two stages in floral evocation

•To initiate floral development, the cells of the meristem must first become competent i.e. capable of responding to floral stimulus (induction).

•A competent vegetative meristem responds to a floral stimulus (induction) to becoming florally determined (committed to producing a flower).

•Once determined, it will flower even after removal of the floral stimulus.

•Competent vegetative shoot (scion) grafted onto a flowering stock will flower.

•Reason: It is capable of responding to floral stimulus present in the stock.

•The grafted scion will fail to flower before attaining competence.

•Reason: Its shoot apical meristem is not yet able to respond to the floral stimulus present in the stock.

•A bud is said to be determined if it progresses to the next developmental stage (flowering) even after being removed from its normal context.

•Thus a florally determined bud will produce flowers even if it is grafted onto a vegetative plant that is not producing any floral stimulus

Demonstration of the determined state of axillary buds in tobacco

•In a day-neutral tobacco, plants typically flower after producing about 41 leaves or nodes.

•If a flowering tobacco plant is decapitated just above the 34th leaf, the axillary bud of the 34th leaf grows out, and flowers after producing 7 more leaves (for a total of 41).

•If the 34th bud is excised from the plant and either rooted or grafted onto a stock without leaves near the base, it produces a complete set of 41 leaves before flowering.

              •Reason: The 34th bud was not yet florally determined.

•If a flowering tobacco plant is decapitated just above the 37th leaf, the 37th axillary bud flowered after producing 4 more leaves in all three situations.

             •Reason:

           ØThe 37th bud was already florally determined.

     ØThe number of nodes a meristem produces before flowering is a function of two factors:

         •Strength of the floral stimulus from the leaves

         •Competence of the meristem to respond to the signal

PHOTOPERIODISM: MONITORING DAY LENGTH

•Photoperiodism is the ability of an organism to detect day length which makes it possible for an event to occur at a particular time of year, thus allowing for a seasonal response.

•Circadian rhythms (Biological clock) and photoperiodism have the common property of responding to cycles of light and darkness.

Plants can be classified by their photoperiodic responses

•The classification of plants according to their photoperiodic responses is usually based on flowering.

•The two main photoperiodic response categories are:

ØLong-day plant (LDPs)

ØShort-day plants (SDPs)

Critical day length

•Flowering in LDPs is promoted only when the day length exceeds a certain duration, called the critical day length, in every 24-hour cycle.

•Promotion of flowering in SDPs requires a day length that is less than the critical day length.

•The absolute value of the critical day length widely varies among species.

                 LDPs flower: Day length > critical day length

                 SDPs flower: Day length < critical day length

Both Xanthium (a SDP) and Arabidopsis (a LDP) flowers under 12 hours of photoperiod.

•Critical day length of Xanthium is 15 hours and it flowers when photoperiod is less than 15 hours).

•Critical day length of Arabidopsis is 11 hours and it flowers when photoperiod is greater than 11 hours.

Since, 12 hours of photoperiod is less than 15 hours and greater than 11 hours, both Xanthium and Arabidopsis can flower.

ØLong-day plants (LDPs):

•Flower only in long days (qualitative LDPs) or flowering is accelerated by long days (quantitative LDPs).

•Flowering promoted only when the day length exceeds critical day length, in every 24-hour cycle.

•LDPs  measure the lengthening days of spring or early summer and delay flowering until the critical day length is reached.

•Ex: Triticum aestivum

ØShort-day plants (SDPs):

•Flower only in short days (qualitative SDPs) or flowering is accelerated by short days (quantitative SDPs)

•Promotion of flowering in SDPs requires a day length that is less than the critical day length.

•SDPs flower in fall, when the days shorten below the critical day length.

•Ex: Chrysanthemum morifolium

•However, day length alone is an ambiguous signal because it cannot distinguish between spring and fall.

•For avoiding the ambiguity of day length signal, plants often couple a temperature requirement to a photoperiodic response.

•Other plants avoid seasonal ambiguity by distinguishing between shortening and lengthening days and are called “dual–day length plants”.

The “dual–day length plants” fall into two categories:

•Long-short-day plants (LSDPs): flower only after a sequence of long days followed by short days. LSDPs,

•Ex: Bryophyllum, Kalanchoe, and Cestrum nocturnum flower in the late summer and fall, when the days are shortening

•Short-long-day plants (SLDPs): flower only after a sequence of short days followed by long days. SLDPs

•Ex: Trifolium repens, Campanula medium, and Echeveria harmsii, flower in the early spring in response to lengthening days.

•Day neutral plants (DNPs): Species that flower under any photoperiodic condition and are insensitive to day length.

   •Flowering in DNPs is typically under autonomous regulation—that is, internal developmental control.

    •Ex: Phaseolus vulgaris, Castilleja chromosa, and Abronia villosa

Plants Monitor Day Length by Measuring the Length of the Night

Night breaks can cancel the effect of the dark period

•The dark period can be made ineffective by interruption with a short exposure to light, called a night break.

•But interrupting a long day with a brief dark period does not cancel the effect of the long day.

•When given during a long dark period, a night break promotes flowering in LDPs and inhibits flowering in SDPs.

•A night break was found to be most effective when given near the middle of a dark period.

Leaf Is the Site of Perception of the Photoperiodic Stimulus

•Treatment of a single leaf of a SDP with short photoperiods are sufficient to cause flowering when the rest of the plant is exposed to long days.

•But, treatment of the shoot apex with short photoperiods don’t induce flowering if the rest of the plant is exposed to long days

•In response to photoperiod, leaf transmits a signal that regulates the transition to flowering at the shoot apex. The photoperiod-regulated processes that occur in the leaves resulting in the transmission of a floral stimulus to the shoot apex is referred to collectively as photoperiodic induction.

Floral stimulus is transported via the phloem

•Once produced, the floral stimulus is transported to the meristem via phloem, and it appears to be chemical in nature.

•Treatments that block phloem transport, such as girdling or localized heat-killing prevent movement of the floral signal.

•The floral stimulus is translocated along with sugars in the phloem and it is subject to source-sink relations.

•An induced leaf positioned close to the shoot apex is more likely to cause flowering than an induced leaf at the base of a stem, which normally feeds the roots.

                                (Images kindly shared by Oxford University Press and are being used for teaching purpose only)