[14-16] - Floods, Seeds and Stomata (Nuhse Lectures)

Question 1

Where is rice predominantly produced, and what are the different forms of rice production?

Answer 1

The vast majority is produced in Asia:

1. IRRIGATED LOWLAND PRODUCTION
   -> 75% of production
   -> 2-3 harvests per year
   -> Requires 3000-5000L water per kg rice
   -> Around 24-30% of global freshwater use annually
2. RAINFED LOWLAND RICE PRODUCTION
   -> No constant supply and/or control of irrigation - more subject to flooding and drought
   -> Usually just 1 harvest per year
   -> More variable yields
3. RAINFED UPLAND RICE PRODUCTION
   -> Fields are never flooded, aerobic soil conditions throughout
   -> Low, variable yields - often used by subsistence farmers with little or no external inputs

Question 2

Briefly summarise the two strategies shown by submergence tolerant strains of rice to cope with flooding

Answer 2

ESCAPE and QUIESCENCE

Escape: rapid growth (internode elongation) to ensure some tissue remains above the water, so that O2 can be fed to the rest of the plant

Quiescence (also known as low-oxygen quiescence syndrome): involves suppression of shoot elongation to preserve nutrients (such as carbohydrates) for as long as possible (up to 10-14 days), with the aim of minimising wasted resources during the submergence period, and then using the conserved carbohydrates to resume growth during desubmergence

Question 3

Explain the ESCAPE strategy in deepwater rice and the molecular mechanism that underpins it

Answer 3

The escape strategy relies on the gaseous plant hormone ETHYLENE

During normal conditions, ethylene escapes into the air; however, submergence prevents it from escaping, causing it to accumulate in plant tissues and induce downstream responses via another hormone (GIBBERELLIN)

Specifically, the ethylene response factors snorkel1/2 are though to promote ABA degradation, thereby increasing GA activity and downstream responses; the Della proteins SLR1 and SLRL1 are also inhibited, allowing GA responses to be activated
-> Evidence that ethylene-induced responses are more complicated: ethylene also induces the ethylene-responsive TF OsEIL1a, which upregulates the GA biosynthesis gene SD1, thereby rapidly increasing GA concentration and promoting internode elongation

GA then appears to act via activation of ACE1 and inhibition of DEC1 to induce internode elongation, which can help rice plants survive flooding, BUT also reduces yield from 6-8t/ha to just 1 t/ha (as spindly plants expend more resources on elongation and thus produce lower yields)

When water rises VERY quickly, rice sometimes cannot grow fast enough for the escape strategy to be effective

Question 4

Explain the QUIESCENCE strategy in rice and the molecular mechanism that underpins it

Answer 4

Submergence-tolerant cultivars (as opposed to deepwater rice) show a range of traits that allow them to withstand complete submergence during flash floods:

Energy maintenance:
-> Minimum elongation
-> Carbohydrate level in stem
-> Optimum fermentation
-> Underwater photosynthesis

Protection:
-> Efficient AOS scavenging
-> Low ethylene synthesis OR sensitivity

The most submergence-tolerant races from India have been collected and screened since the 1970s.
-> The FR13A landrace is submergence tolerant (and shows dominance)
-> The Sub1 locus was identified in 2006, and accounts for around 70% of variation in submergence tolerance
-> This is conferred by the Sub1A-1 gene, and ethylene response factor (the Sub1A gene is very similar to SNORKEL1/2 despite having the opposite effect)

Pathway:
Ethylene activates SUB1A, which STABILISES SLR1 + SLRL1 (which then inhibit GA responses) and also inhibits ethylene (negative feedback)

Question 5

What was the variety of rice that was bred to be high-yielding but also submergence tolerant?

Answer 5

Swarna-Sub1 rice was developed in the 2000s by repeatedly crossing the submergence tolerant (but low yielding) Sub1 variety with the high yielding (but flood-prone) Swarna variety, until a variety was produced with a very similar genome to Swarna, but retaining the Sub1 gene [Marker-assisted selection, MAS]

This allowed the Sub1 trait to be rapidly introgressed into Swarna without the need for GM (which would have required regulation and been viewed sceptically by farmers)

The Swarna-Sub1 variety now accounts for over 25% of rice planted in India -> popular because high-yielding, flooding-insensitive, AND produced grains with a more favourable colour for rituals

Question 6

What allowed the ethylene signalling pathway to be discovered?

Answer 6

The TRIPLE RESPONSE:

• The addition of ethylene induces a distinct Triple Response
  -> an exaggerated Apical Hook
  -> a short root
  -> a short hypocotyl

By screening for mutants in this pathway and these specific responses, it was possible to identify genes involved in ethylene responses

Question 7

What was the first ethylene response mutant to be discovered, and what did it reveal about the underlying mechanism?

Answer 7

etr1 (EThylene Response mutant)

-> A mutation in a gene similar to prokaryotic two-component sensors - previously, no such response regulators had been discovered in eukaryotes (a similar one has since been discovered in tomatoes, never-ripe)

Mechanism:
-> In total, there are 5 ethylene receptors in Arabidopsis
-> Etr1, Etr2, Ers1 (ethylene response sesnor 1), Ers2 and Ein4 (ethylene-insensitive 4)
-> These all reside in the ER and can form heteromeric complexes
-> Need to look at second-most-important mutant to fully piece together the mechanism

Question 8

What was the second key ethylene response mutant that allowed more of the underlying mechanism to be understood?

Answer 8

ctr1 (constitutive triple response)

-> Mutation in a Raf-like kinase
-> CTR1 is a negative regulator of ethylene signalling

Whole pathway:
-> In the ABSENCE of ethylene, the receptors (Etr1 etc.) are active, and activate CTR1, which in turn inhibits downstream ethylene responses
-> However, in the PRESENCE of ethylene, the receptors bind it and are inactivated, meaning CTR1 is also inactive (therefore, ethylene responses are NOT inhibited, and can be induced

Note on mutants:
-> A missense mutation at the binding site of one of these receptors prevents ligand binding and makes the receptor insensitive to ethylene - this can lead to suppression of responses even in the presence of ethylene
-> Meanwhile, disruptions in the regulatory domains of at least three ethylene receptors can lead to receptor INactivation, even in the absence of ethylene, leading to a constitutive ethylene response
-> For a similar reason, LoF mutations in CTR1 lead to a constitutive ethylene response, as the pathway cannot be inhibited

Question 9

What was the next protein mentioned after the ethylene receptors and CTR1, which explains how CTR1 is linked to downstream responses?

Answer 9

EIN2 - a positive regulator of ET signalling

LoF mutants in EIN2 are ethylene INsensitive, showing that it normally has a positive role. It has 12 membrane-spanning domains and was known to be downstream of CTR1, but how this signal transduction occurred was unknown until 2012

-> Active CTR1 phosphorylates and targets the C-terminal domain of EIN2 for degradation at the 26S proteasome, along with the TFs EIN3 and EIL1
-> When CTR1 is INactive (either due to ethylene binding at the receptor, or due to LoF mutations in CTR1), EIN2 is not phosphorylated, and the C-terminal region (along with EIN3/EIL1) is cleaved, translocates to the nucleus and promotes ethylene responsive genes via ERF1 (a member of the ERF TF subfamily)

Question 10

Summarise the key steps in Ethylene Biosynthesis

Answer 10

1. Methionine is converted to S-adenosylmethionine (AdoMet) via AdoMet Synthase
2. AdoMet is converted to ACC (1-aminocyclopropane-1-carboxylic acid) via ACC Synthase (ACS)
3. ACC is converted to ETHYLENE via ACC Oxidase (ACO)

Question 11

Explain which enzyme in the Ethylene Biosynthesis Pathway is tightly regulated (and was explained in the lecture)

Answer 11

ACC Synthase (ACS):

-> ACS is highly unstable, and is constantly synthesised and degraded again, keeping ethylene levels low under normal conditions
-> Phosphorylation of the ACS C-terminus can prevent interaction with the 26S proteasome, thus stabilising ACS
-> This can happen in response to wounding or pathogen attack (via MAPK) or abiotic stress such as cold (via CDPK)

This is how ethylene levels are increased in response to biotic or abiotic stresses such as these

Question 12

Summarise the wide range of whole-plant processes in which ethylene is involved

Answer 12

-> Shoot + Root Elongation
-> Flooding responses (e.g., aerenchyma formation, leaf epinasty, deepwater rice elongation)
-> Pathogen Responses
-> Reproductive Development (e.g., sex determination in cucumbers, petal senescence, fruit ripening)

This is demonstrated by the fact that ethylene blockers (e.g., silver thiosulfate and CACP) promote longevity in cut flowers; also the fact that ethylene production peaks before ripening in climacteric fruit, and that inhibiting ethylene synthesis can slow down fruit ripening

Question 13

Very briefly describe how fertilisation occurs in plants

Answer 13

Double fertilisation:
-> One sperm cell fuses with the egg cell to form the zygote, while the other fuses with the nuclei of the central cell to form the triploid (3n) endosperm

(The developing embryo remains tethered to the plant by a suspensor)

Question 14

When does seed maturation begin, what are the key changes that occur, and which hormone plays a central role in this process?

Answer 14

It begins after the completion of embryo development (i.e., once cell division stops)

The four key changes are:
-> Transition from maternal to filial control (i.e., the embryo takes over control of development from the maternal tissue)
-> Accumulation of storage compounds (e.g., starch, proteins, fat - depends on the plant/seed type)
-> Establishment of primary dormancy (the first step of preparing the seed for desiccation)
-> Acquisition of desiccation tolerance (LEA genes established), then desiccation

In terms of fresh weight:
STAGE 1: Weight remains constant but rapid cell division and differentiation are occurring
STAGE 2: Water weight increases, peaks, then starts to decrease while dry weight increases (cell expansion and accumulation of storage compounds during this time)
STAGE 3: Water weight decreases as seeds prepare for desiccation

ABSCISIC ACID (ABA) plays a central role in this process

Question 15

What are the key changes/phases that a seed undergoes following maturation, and how does this vary between different types of seeds?

Answer 15

Eventually, germination will begin, but this depends on the seed type:
-> Orthodox seeds remain dormant for a while, then lose dormancy but remain quiescent (a resting state with low metabolism), until suitable environmental conditions allow germination
-> Evolution has selected for some seeds to remain dormant for longer (so that, even if a catastrophic event kills all the germinated seeds, the dormant ones are not lost)
-> Recalcitrant (“unorthodox”) seeds are NOT tolerant to drying out and do not have long term viability, they must germinate soon after maturation [e.g., mango, lychee, avocado, cocoa]
-> Vivipary occurs when induction of dormancy fails and immature seeds germinate while on the mother plant (normal for mangroves, mutants in other species)

In PHASE 1 of germination (imbibition):
-> seedling swells, fresh weight increases
-> this occurs by simple physical uptake of water

In PHASE 2 of germination (lag):
-> intense metabolic activity
-> mitochondrial activation, protein synthesis, gene expression, hydrolysis of cell walls, breakdown of storage products, etc.

In PHASE 3 of germination:
-> continued water uptake
-> weakening of endosperm cell walls
-> seed coat + endosperm rupture; radicle emergence due to cell expansion

Question 16

State which signals control germination, and how mutant maize helped the main hormone inhibiting germination to be identified

Answer 16

Germination involves integration of multiple signals:

Inhibitory factors (e.g., high temperature, drought) signal through ABA

Promoting factors (e.g., light, cold stratification) signal through GA

The vp14 (viviparous14) maize mutant was useful in discovering this:
-> this mutant undergoes much more rapid water loss than the WT during germination
-> ABA levels also much lower in mutant than WT
-> the VP14 protein was found to be an NCED (9-cis-epoxy-carotenoid dehydrogenase) protein, catalysing a crucial step in the pathway by which the ABA precursor Xanthoxin is produced from Zeaxanthin in the plastid
-> Therefore, the vp14 was deficient in ABA synthesis, explaining the low ABA levels and implying that ABA is a negative regulator of germination

In WT plants, NCED genes are induced by stress (e.g., drought, or within 30 minutes of leaf detachment) and during seed maturation, where it is responsible for processes contributing to seed dormancy, accumulation of reserves, and desiccation tolerance

Question 17

State where GA was first identified, explain what is notable about the GA biosynthesis pathway, how it interacts with ABA and the role of GA in germination

Answer 17

GA was first discovered as a fungal compound that promotes stem elongation in Bakanae disease

The early steps in the GA biosynthesis pathway (e.g., the CPS and KS-catalysed steps in the proplastid, and the KO and KAO-catalysed steps in the ER membrane) are generally controlled by single enzymes (with KO leading to severe phenotypes)
-> For example, the ga-1 mutant of tomato is defective in CPS, and has a ~0% germination rate, but can be completely rescued by supplementing GA

The later stages in the cytoplasm, on the other hand, are catalysed by multiple isoforms (e.g., GA 13-hydroxylase, GA 20-oxidase, GA 3-oxidase), some of which exhibit tissue-specific expression (meaning it may be possible to suppress GA only in the stem)

The balance of ABA and GA activity determines the switch from dormancy -> nondormancy -> germination
(ABA signalling, via regulators such as ABI5, ABI3 and ABI4, promotes dormancy)
(GA signalling, via the TF GAMYB, promotes the transition from dormancy to non-dormancy, and thence to germination)

This balance is affected by environmental factors (e.g., sunlight promotes germination, as it activates Phytochromes, which promote degradation of PIF, meaning PIF no longer inhibits GA and promotes ABA; meanwhile, nitrate also promotes degradation of ABA and therefore germination)

In germinating monocot seeds, GAs moves from the embryo (where they are synthesised) to the endosperm via the scutellum
-> they then diffuse into the aleurone layer, where they induce cells to synthesise and secrete hydrolases (such as a-amylase) into the endosperm
-> this leads to breakdown of starch and other macromolecules
-> the resulting solutes can then be absorbed by the scutellum and transported to the growing embryo

Question 18

State why germination is important in beer brewing

Answer 18

In beer brewing, it is desirable for as many of the seeds to have germinated as possible, so that they can produce sugars, before they are roasted and yeast added to ferment the sugars to alcohol

For this reason, seeds are often sprayed with GA to ensure they have all germinated

Question 19

From a practical/agricultural perspective, what is the significance of seed dormancy and germination?

Answer 19

For domestic crops, low dormancy and uniform germination are generally desired (opposite of what is selected in the wild, therefore negative breeding)

This is because high dormancy increases storage costs (due to after-ripening of seeds) and a lower percentage of germinated seeds in the field

However, a certain level of dormancy is still required to prevent pre-harvest sprouting (PHS)
-> $1 billion is lost annually through PHS in wheat
-> PHS resistance has been lost in white wheat preferred by customers (alongside browning bran colour, phenolic compounds and strong flavour)

Question 20

What is the final key integrator of ABA and GA signalling mentioned at the end of the lecture?

Answer 20

MOTHER-OF-FT-AND-TFL1 (MFT) is a central integrator in the signalling “tug-of-war” between ABA and GA

It appears to repress seed germination under FR light by modulating phytohormone responses

It is activated downstream of ABA via ABI5, and inhibited when GA is active due to inhibition of DELLA proteins

Question 21

Explain how the quality of wheat is measured, and how this relates to the topics discussed in Lecture 16

Answer 21

-> Wheat grains milled and cooked with water to form “porridge” into which a steel ball is dropped, and the time taken for the ball to settle measured
-> Hagberg Falling Number (HFN) is an indirect measure of bread properties (low HFN indicates poor quality, difficult to slice due to sticky crumb, 28% of wheat not sold for bread production due to low HFN)
-> It is not fully understood which factors determine the HFN of wheat, but cool wet summers that promote PHS are thought to reduce the HFN
-> More research into these factors

A study by White et al (2022) identified six QTL for HFN, and argued that this supports the possibility of genetically improving the HFN of wheat in the future

Question 22

Describe how current climate models differ from those first devised in the 1960s

Answer 22

The first climate models were far more limited by the technology of the time, and were simple models aiming to understand the physics of earth’s surface

The most recent climate models, Earth System Models (ESMs) are able to account for far more factors - including physical, chemical and biological factors - to make predictions

Question 23

Explain the key ways in which plants affect the physics of earth and the climate, and the ways in which recent climate models have tried to account for this

Answer 23

Plants and vegetation impact the climate in several key ways:

BIOCHEMISTRY:
- Carbon flux
- Sequestration
(Especially in mature and tropical forests)

BIOGEOPHYSICS
- Albedo (the brightness of the land surface) is affected by vegetation, e.g., forests are darker than bare soil, boreal forests are much darker than snow-covered soil, early growing season crops are lighter
- Evapotranspiration (evaporative cooling is greater in crops than forests, and greater in tropical than boreal forests)

BIOGEOGRAPHY
- Land use change due to agriculture
- Deforestation and reforestation
- Fire

One of the most sophisticated recent ESMs from the Geophysical Fluid Dynamics Laboratory at Princeton (GFDL) accounted for the following major factors:

PLANT GROWTH
- Photosynthesis and respiration (CO2, H2O, light, temperature)
- Carbon allocation to leaves, wood, roots, storage

PLANT FUNCTIONAL DIVERSITY
(Tropical evergreen/coniferous/deciduous trees, warm/cold grasses etc)

DYNAMIC VEGETATION DISTRIBUTION
- Competition between functional plant types
- Natural fire disturbance (affected by drought and biomass)

LAND USE
- Cropland, pastures, natural and secondary lands
- Conversion of natural and secondary lands and abandonment
- Agricultural and wood harvesting and resulting fluxes

Question 24

State the equation for water use efficiency, give the approximate WUE values for three different types of plants, and state the reasons why it is so low

Answer 24

WUE = mol CO2 fixed by photosynthesis/H2O lost via transpiration

C3 plants: 1/400
C4 plants: 1/150
CAM plants: 1/50

Low because:
- Concentration gradient driving water loss is about 50X that driving CO2 uptake
- CO2 diffuses 1.6 times more slowly through air compared to water
- Lost water simply has to pass through the stomatal pore, whereas CO2 must cross multiple layers, including the plasma membrane, cytoplasm and chloroplast envelope

Question 25

What is meant by the “Optimal Stomatal Theory” and why is this an important question in the context of climate change?

Answer 25

Stomata present a dilemma for plants - opening more stomata enables greater the CO2 uptake for photosynthesis; however, it also increases the water penalty, as significantly more water will be lost via transpiration

Optimal stomatal theory suggests that plants will try to optimise stomatal opening for maximum carbon gain with minimal water loss

-> The important question is how this will be affected by increasing atmospheric CO2

CO2 is a limiting factor in photosynthesis, so increasing [CO2] promotes carbon fixation by Rubisco, leading to increased sequestration [this effect is called carbon fertilisation]

However, as [CO2] increases, stomatal conductance decreases, improving WUE but limiting carbon sequestration

-> Important to determine the NET effect of increasing CO2 on carbon storage

Question 26

Describe the experiments that have been carried out to try and determine the effect of increasing [CO2] on stomatal aperture and photosynthesis [[and state the conclusion of the opinion paper highlighted in the lecture]]

Answer 26

Free Air CO2 Enrichment (FACE) experiments provide the most realistic measurements of CO2 impact

By pumping CO2 into a designated circle of crops or vegetation without enclosure, it is possible to study the effects of elevated CO2 in otherwise natural conditions

-> Such studies have been ongoing for over 30 years, and meta-analyses have provided significant insight

[[The terrestrial biosphere overall has shown an increase in leaf-level photosynthesis, water use efficiency, and gross primary production (GPP) proportional to the increasing concentration of CO2 in the atmosphere. Cernusak et al (2019) also argued that the improving WUE could partially mitigate the impact of drought stress on vegetation -> HOWEVER, it cannot be assumed that this trend will continue as CO2 continues to increase – saturating photosynthesis, or maximisation of leaf area per unit ground area may limit further increases in GPP.]]

More recently, the Amazon Face project has been established to measure the impacts of rising CO2 in the Amazon Rainforest, using a similar principle to other FACE experiments

Question 27

State the main cues to which guard cells respond

Answer 27

CO2, Ozone, Drought, ABA; Light, Low CO2, Humidity

Question 28

How do the stomata affect leaf temperature?

Answer 28

The more stomata are open, the cooler the leaf, due to the cooling effect of water evaporation

Francis Darwin proposed that leaf temperature could therefore be used to estimate the level of stomatal opening

Based on this principle, thermal imaging has been used to identify Open Stomata (OST) Mutants, as their leaves are visibly cooler than wild-type plants

Question 29

Describe and explain the important OST mutants mentioned in the lecture, and what they reveal about stomatal regulation in plants

Answer 29

Firstly, note that mutants impaired in ABA synthesis (such as aba2-13) or ABA signalling (such as ost1-4) show reduced stomatal closure in low humidity compared to WT plants, and are therefore cooler (as seen by thermal imaging)

OST1:
ost1 mutants were identified this way, and allowed OST1 (also known as SnRK2.6) to be identified -> a kinase expressed in guard cells and vascular tissues, which is required for stomatal closure and belongs to a family of highly conserved kinases that also includes yeast SNF1 and mammalian AMPK

ABI1-1:
abi1-1 mutants are ABA insensitive in all responses (germination and root growth not inhibited by ABA, guard cells not ABA-responsive)
ABI1 encodes a PP2C protein phosphatase (PP2Cs are important, see next FC)

PYR/RCAR:
PYR/RCAR are ABA receptors which are also necessary for ABA responses, including guard cell closure
-> pyr/rcar mutants are insensitive to ABA and germinate on ABA-containing medium

SLAC1:
slac1 mutants do not show stomatal closure in response to ozone (as well as CO2, ABA, humidity change, light/dark transitions, Ca2+ etc.)
-> SLAC1 is a slow anion channel in guard cells

Question 30

Explain the underlying biochemical mechanisms underpinning the function of guard cells

Answer 30

PP2C phosphatases (e.g., ABI1) interfere with the activity of SnRK2 kinases:
-> in the absence of ABA, PP2Cs bind and sequester SnRK2 kinases
-> however, when ABA binds the PYL/PYR receptor, it induces a conformational change, causing PYL to mimic the activation loop of SnRK2, and then displace SnRK2 from PP2C
-> SnRK2 is now no longer inhibited and is released to activate downstream targets of ABA

Once SnRK2 is released from PP2C inhibition, it activates and inhibits many downstream transcription factors, such as AREBs, via phosphorylation.
-> Importantly, subclass III SnRK2s, such as SnRK2E (OST1) also phosphorylate and activate membrane proteins such as SLAC1 and KAT1, which both allow efflux of anions
-> This efflux of anions leads to reduced turgor pressure, causing the stomatal pore to close

Meanwhile, SnRK2s also phosphorylate the respiratory burst oxidase AtrbohF, which produces superoxide (O2-) which then activates internal Ca2+ channels, releasing Ca2+ into the cytoplasm
-> Ca2+ then activates calcium-dependent kinases (CDPKs), which further phosphorylate and activate SLAC1, and also deactivate the proton pump OST2 via phosphorylation

The mechanism by which CO2 specifically causes stomatal closure is not entirely understood
-> it appears to rely on OST1 (meaning OST1 integrates CO2 and ABA signals) and SLAC1
-> It requires the carbonic anhydrases CA1 and CA4, suggesting that only bicarbonate can directly modulate SLAC1 activity
-> a guard cell-specific kinase (HT1) acts as a negative regulator of CO2 responses -> ht1 mutants are CO2 hypersensitive but respond normally to ABA

Question 31

Describe the effect of CO2 on stomatal development

Answer 31

Stomatal density on the leaf surface is tightly regulated during plant development, via spatial control of asymmetric cell division

Higher [CO2] decreases the density of stomata (specimens from the pre-industrial age show that stomatal density was higher when CO2 levels were lower)!