[17-19] - Plant Architecture and Cells

Question 1

State the equations for Primary Production and Yield Potential

Answer 1

Primary Production (Pn) = St x ei x ec / k

S = annual integral of incident solar radiation

ei = efficiency with which that radiation is intercepted by crops

ec = efficiency with which intercepted radiation is converted into biomass

k = energy content of plant mass

Yield Potential (Yp) = n x Pn

n = harvest index (efficiency with which biomass is partitioned into the harvested product)

Question 2

What was one of the major problems with conventional varieties of crops before the Green Revolution, and how did the Green Revolution improve this?

Answer 2

Nitrogen fertilisation is essential to increase grain yield, but also promotes leaf and stem elongation

This results in an increase in plant height, which increases the risk of plants falling over due to excessive height, leading to fungal infections, pre-harvest sprouting and other issues which led to yield losses

The Green Revolution introduced semi-dwarf varieties, the short stature of which conferred lodging resistance under high N fertilization

Conventional varieties of wheat and rice:
n = 0.3 (30% grain, 70% straw);
total biomass = 10-12 t/ha;
maximum yield potential = 0.3x12 = 4t/ha

Green Revolution Varieties:
n = 0.5 (50% grain, 50% straw)
total biomass = 20 t/ha
maximum yield potential = 10 t/ha (more than double)

This turned countries from grain importers into grain exporters

Question 3

State some of the processes in which Gibberellin is involved

Answer 3

• Growth
• Seed germination
• Promote flowering
• Promote sex determination in some species
• Promote fruit growth

Question 4

Explain the phenotypic effects of mutations in different GA biosynthesis genes

Answer 4

LoF mutants of enzymes catalysing the early stages of GA biosynthesis (e.g., CPS or KS) are severely dwarfed and are too small to be agriculturally useful, as these steps are catalysed by one or very few genes, so mutants have severely reduced GA levels.

However, the enzymes catalysing the later stages are encoded by multiple genes, some of which are expressed only in certain parts of the plant
-> For example, sd1 variety is mutated in a GA20ox (GA 20-oxidase) gene that is expressed in shoots, but not seeds, which leads to increased grain yields

Growth and yield can also be optimised by tissue-specific GA catabolism: overexpression of the GA deactivating enzyme GA2ox under a GA3OX promoter (which acts only in internodes) resulted in a new variety of dwarf plants which also showed high yields

Question 5

How are the genes responsible for GA biosynthesis and deactivation controlled?

Answer 5

These pathways (like many biosynthetic pathways in plants) are very tightly regulated, with most genes expressed in a cell-specific manner

Some examples of regulation:
- Auxin upregulates GA synthesis by promoting 20-oxidase and 3ß-hydroxylase
- Temperature and light regulate GA3ox
- Active GAs downregulate their own synthesis and upregulate their own deactivation (negative feedback) -> e.g., by inhibiting GA3ox, GA20ox and promoting GA2ox

Question 6

What is the analogy used for how GA activates plant responses?

Answer 6

A car stuck at a roadblock:
- The car is the transcription factors which can activate target genes
- DELLA proteins are the roadblock preventing them from promoting transcription
- Active GAs remove the roadblock (DELLA) proteins, allowing the car (TFs) to act

Question 7

Briefly describe how three different types of GA mutant can be distinguished phenotypically

Answer 7

GA Biosynthesis mutants -> GA-sensitive dwarfs (i.e., dwarf in absence of GA but rescued by adding GA)

GA LoF Response mutants with signalling defects -> GA-insensitive dwarfs (i.e., dwarf in presence OR absence of GA)

GA GoF response mutants with constitutive signalling -> Slender (i.e., grow large in presence OR absence of GA, like a WT stimulated by GA)

Question 8

Explain the GA signalling pathway and the mutants that help to elucidate it

Answer 8

GA binds a GA RECEPTOR (GID1 = GIBBERELLIN INSENSITIVE DWARF1)
-> Rice gid1 mutants are dwarf-like and are NOT rescued by GA as they cannot respond to it

When GA binds the GID1 receptor, it causes a conformational change, whereby the N-terminal extension switch folds over the GA binding site. This conformational change allows the complex to be recognised by the DELLA proteins (members of the GRAS family), which interact and are subsequently degraded, preventing them from suppressing target TFs

Note that the DELLA domain is required for GID1-binding, and mutants with deletions of the DELLA domain (e.g., gai1) are GA-insensitive dwarfs as DELLA cannot bind the GID1 receptor and is therefore stabilised and constantly represses target TFs, while LoF DELLA mutations (e.g., SLENDER1) result in excessive elongation as DELLA is unable to repress its target genes

SLEEPY/GID2, on the other hand, encode components (specifically F-box proteins) of the SCF E3 ubiquitin ligase complex, which is responsible for ubiquitinating DELLA proteins such as RGA and targeting them for degradation at the 26S proteasome
-> LoF mutations in these result in GA-insensitive dwarf mutants
-> GoF mutations in these genes (e.g., sly1-d) result in increased GA signalling via enhanced ubiquitination of DELLA proteins

Question 9

Which wheat mutant was extremely important during the Green Revolution, and what is the biochemical basis for it?

Answer 9

REDUCED HEIGHT1 (RHT1) encodes a DELLA protein

-> The dwarf allele rht1 lacks the DELLA domain and is resistant to proteolysis (i.e., DELLA protein has increased stability)

-> this results in a semi-dwarf phenotype, which was key during the Green Revolution

Question 10

What is one well-understood example of a GA response and signalling?

Answer 10

ETIOLATION - a form of photomorphogenesis

In etiolation, plants undergo phenotypic changes in response to darkness, including elongated shoot/hypocotyl growth and unopened leaves; GA-deficient mutants (e.g., the ‘na’ mutant in peas) do not show this normal dark growth pattern, but can be rescued with exogenous GA

PIF3 and PIF4 are TFs which promote growth-related genes, leading to etiolation in response to darkness; in the absence of GA, DELLA proteins bind and inhibit PIF3/4

However, in the presence of GA, GA promotes DELLA degradation, so PIF3/4 are released and can regulate target genes, for example by interacting with HISTONE DEACETYLASE15 to promote histone acetylation, or inhibiting the binding of TCP4-like TFs to SAUR genes

Question 11

How significant is plant architecture as a target for domestication (and which species was used to demonstrate this)?

Answer 11

Architectural Traits (e.g., form and height) have been significantly altered by domestication and improvement:

E.g., Brassica oleracea (wild cabbage) gave rise to broccoli, kale, kohlrabi, cauliflower, cabbage and brussels sprouts
-> Also, maize vs wild teosinte (architectural changes allowed much denser crop production)

Understanding the genetic basis and physiological consequences of these changes could aid crop breeding in the future -> this requires understanding differential growth, as this fundamentally determines differences in morphology and architecture

Question 12

Summarise the brief section on ‘Biophysics of Plant Growth’

Answer 12

For a cell to grow or expand, the turgor pressure inside must be high enough to drive expansion of the cell wall (which then reduce the turgor pressure, allowing more water to enter)

Note: in reality, this occurs as one continuous process rather than distinct steps

Question 13

Describe the structure, synthesis and organisation of plant cell walls

Answer 13

Plant cell walls are complex (encoded by around 10% of the genome) and consist of a network including many components, e.g., pectin, lignin, and the main component - ß-cellulose

ß-cellulose, unlike a-cellulose, consists of monomers which are flipped 180 degrees relative to their adjacent monomers, allowing organisation of chains into very strong, insoluble microfibrils, held together by H-bonding between chains

Cellulose is synthesised at the plasma membrane (and simultaneously secreted out to form fibrils) by a very large, rosette-like cellulose synthase complex, which produces multiple chains simultaneously

The structure of the microfibrils is determined by the organisation of catalytic sites within the rosette structure in the plasma membrane
-> the ORIENTATION of the innermost cellulose layers is determined by that of the microtubules, which directly attach to cellulose synthase complexes and can reorientate them
-> this is crucial for plant cell expansion, as the direction of cell expansion is always perpendicular to the orientation of the inner layers of cellulose microfibrils

Question 14

How can the orientation of plant cell expansion be externally controlled?

Answer 14

Plant hormones can induce re-orientation of the microtubules (as demonstrated by transfer of cells from ethylene to GA -> 90 degree change in MT orientation)

This in turn controls the orientation of plant cell expansion:
-> GA promotes transverse MT orientation (and thus vertical shoot growth, leading to long, thin plants)
-> Ethylene promotes longitudinal MT orientation (and thus wider and shorter architecture) - can see the effect of this in ctr1 mutants, which show a constitutive ethylene response due to a mutant regulator, and are shorter than WT

Question 15

How can the properties of the plant cell wall (i.e., rigidity) be measured (and what are the group of factors which are known to promote loosening?

Answer 15

An EXTENSOMETER is used to measure plant cell wall ‘creep’ when a constant force is applied - conditions can then be changed and their effect on plant cell wall expansion observed (e.g., much faster expansion at pH 4.5 than pH 7)

To identify the factors responsible for controlling cell wall rigidity, heat-inactivated stems were used as a control, then different fractions of homogenized tissue were added -> successfully identified factors promoting expansion (EXPANSINS)

Question 16

Explain the evidence disproving the older model of plant cell wall expansion, and explain the newer model which is now preferred

Answer 16

Previously, it was thought that cellulose microfibrils were cross-linked by glycans (e.g., xyloglucan), and that cell wall loosening was brought about by wall-loosening enzymes

However, some observations of Arabidopsis xxt1/xxt2 double mutants (which lack detectable xyloglucan) could not be explained by this model
-> Despite the hypocotyls being 30% weaker than WT, the cell walls were LESS extensible in creep assays, and were less sensitive to expansins

Meanwhile, xyloglucan-specific endoglucanase did not weaken the walls of WT (despite digesting much of the xyloglucan), and a mixture of cellulose-specific and xyloglucan-specific endoglucanases ALSO did not loosen the cell wall
-> ONLY an enzyme able to cut BOTH xyloglucan and cellulose could loosen the cell wall

This led to the Biomechanical Hotspot concept:
-> Wall extension is controlled at limited sites of close contact between cellulose microfibrils, mediated by xyloglucan chains
-> The xyloglucan chains may be intertwined with cellulose chains at these hotspots, forming an amalgam which can only be properly digested by an enzyme with both cellulase and xyloglucanase activity
-> Alternatively, xyloglucan may form a tight monomolecular junction between multiple cellulose microfibrils, rendering it inaccessible

Question 17

How might our understanding of plant cell walls and the enzymes regulating their expansion be applied to improve crop yields?

Answer 17

Previous attempts to improve wheat yield via increasing individual grain weight have been unsuccessful, as improvements have been offset by reductions in grain number (negative association)

However, expansins are a promising target to overcome this:
Expansins are a relatively broad enzyme family, consisting of four sub-families (a-exp, ß-exp, exp-like A and exp-like B), and are known to play a role in many aspects of plant development, such as germination, leaf growth, stomatal closure, among others. This makes them a promising target for crop improvement – for example, in one experiment by Calderini et al. (2021), a root expansin (TaExpA6) was expressed in wheat using a grain filling-specific promoter (PinB), aiming to target a stage after grain number is determined. This proved successful in increasing grain size and yield, with the best-performing transgenic line achieving a yield 12% greater than the wild-type, and 11% better in field trials(!!!). The authors consequently argued that this was a proof of concept for the tissue- and time-specific overexpression of expansins as a means of improving grain size without sacrificing grain number.

Another example of the potential for crop improvement targeting expansins was demonstrated by Minoia et al. (2016) who showed that loss-of-function (LoF) mutations in the tomato expansin gene SlExp1 led to firmer, later ripening fruits with an improved shelf life.

Question 18

How can the canopy be a target for improved Primary Production (sometimes counterintuitively)?

Answer 18

A closed canopy (i.e., one in which all the light is intercepted by leaves and none hits the ground) is not necessarily optimised for maximum interception of solar radiation (Ei)

If all of the leaves are at wide angles to the shoot (i.e., as opposed to more vertical), then those at the top will intercept far more light than they can utilise for photosynthesis, whereas those lower down will receive far less light

The efficiency of light capture in such a canopy can be improved by reducing the leaf angle of leaves at the top (i.e., ensuring the top leaves grow more vertically), thereby allowing leaves at all levels to intercept a moderately high amount of light

Overall, this allows greater Photosynthetic Photon Flux Density (PPFD) than flat leaves/branches

Question 19

What are brassinosteroids, what is the most active of them, and what are their roles in plants?

Answer 19

Brassinosteroids are a group of steroidal phytohormones found in plats, with brassinolide being the most active

They have roles in many important plant processes, including cell elongation, pollen tube growth, seed germination, differentiation of vascular tissues/root hairs, and stress tolerance

(They were first discovered when it was found that treating beans with pollen extract caused stem and cell elongation)

Question 20

State the main BR biosynthesis mutants which helped to elucidate the pathway

Answer 20

Mutants in:

DWF1/LKB/BRD2

DWF4

DET2

CYP85A1,2

CYP85A3

are severely dwarfed, as they lead to BR-deficiency

Question 21

Explain how BR is sensed/perceived, and how this leads to downstream responses

Answer 21

BR binds a BR Receptor Complex:

• The main receptor is BRI1 (bri1 mutants are BR-insensitive dwarfs)
• BAK1 is a co-receptor (in the absence of BR, BAK1 is prevented from interacting with BRI1 by both the ecto- and endo-domains
• BKI1 is an inhibitor which physically interacts with the BRI1 receptor on the cytoplasmic side

When BR binds the complex, phosphorylation of BKI1 causes dissociation, which then allows the co-receptor BAK1 to interact -> BAK1 and BRI1 then phosphorylate each other, producing an active kinase domain which targets BIN2:

• Normally, in the absence of BR, BIN2 is phosphorylated and activated, and inhibits target TFs via phosphorylation
• When the receptor is activated by BR, the active kinase domain phosphorylates (and activates) BSKs
  -> BSKs then phosphorylate (and activate) BSU1
  -> BSU1 then DEphosphorylates (and INactivates) BIN2
  -> Target TFs (most importantly BZR1 and BES1) are no longer phosphorylated, and are free to promote and inhibit a range of genes leading to downstream BR responses

Question 22

Explain some of the responses induced by the targets of BR signalling, and why this pathway is difficult to target via GM

Answer 22

BZR directly promotes shoot branching by upregulating target genes

It also indirectly inhibits CUCs (which control organ boundaries)

BZR also forms complexes with ARF6 and PIF to upregulate PRE, which then inhibits IBH1 (a gene responsible for aging) and PAR1 (a SAS inhibitor) while indirectly upregulating HBI1 (which has roles in photosynthesis and CELL ELONGATION!)

Because the targets of BR (e.g., BZR) are so central in this complex signalling pathway, knocking out BR will lead to many different effects, including unfavourable ones
-> Ideally, we want to alter some of these responses in a more targeted way, while leaving most of these signals intact

Question 23

What is notable about the rice Brassinosteroid Biosynthesis mutants shown in images on the slides?

Answer 23

There is phenotypic variation between different mutants (e.g., some more severe than others), but even in the least severe (e.g., osdwarf4-1), the branching angle is noticeably smaller, with more erect leaves than the WT rice

Question 24

Explain what was observed in a field-grown osdwarf4-1 mutant

Answer 24

When grown at high densities, the osdwarf4-1 mutant (with its erect leaves caused by BR deficiency) show increased biomass production and increased grain yield compared to the WT

Additionally, the grain yield of the mutant rice was unaffected by additional fertiliser

Question 25

Name the rice putative Brassinosteroid receptor gene mutated in several dwarf varieties, and describe how well ones of these mutants performed in field trials

Answer 25

A rice (Oryza sativa) ortholog of BRASSINOSTEROID INSENSITIVE1, OsBRI1

Mutations in this gene give rise to the d61 dwarf phenotype in rice, and several different point mutations are known to give slightly different phenotypes

The weakest of these alleles, d61-7 was tested in paddy field trials:
-> At high planting density, d61-7 exhibited 35% greater biomass than the WT, and the grain yield continued to increase with planting density (unlike that of the WT), suggesting that the mutation confers more effective biomass production
-> HOWEVER, this does not result in an increase in overall yield, as decreased grain size offsets the increase in biomass, even at the highest planting densities

Question 26

What do grafting studies suggest about the nature of BR as a signal?

Answer 26

Grafting in peas has shown that WT roots CANNOT rescue BR-deficient shoots that have been grafted

This demonstrates that, unlike GA, BR is not transported long distances throughout the plant, but rather is made and acts locally
-> This could be exploited for tissue-specific overexpression

Question 27

How does modification of leaf angle in maize link back to the early lectures of this module?

Answer 27

Comparing maize with teosinte reveals an example of a genetic bottleneck in domestication (as certain alleles are selected during domestication, while many others are discarded) -> there is likely to be great diversity among wild ancestors of crop maize (i.e., teosinte) that could potentially be exploited today for crop improvement (e.g., improving branching architecture of maize)

Question 28

What genes and mutations can affect leaf angle in maize?

Answer 28

Mutations removing the ligule (e.g., liguleless1/lg1 and lg2) result in a very upright leaf angle

Mutations affecting the size of the auricle (e.g., alterations in BR signalling) can also affect leaf angle
-> Application of exogenous BR can increase auricle size and leaf angle, while loss of BR has the reverse effect

The thickness of the leaf at the midvein/midrib can also affect leaf angle (e.g., drooping leaf/drl mutants lack a midrib, and thus have floppy leaves with wider leaf angles)

Note that all of these mutants also have other unfavourable effects on plant stature, floral patterning etc. due to the complexity of the pathway

Question 29

How can teosinte be used to improve the modern maize crop (example at end of lecture 19)? Discuss the genetics, biochemistry, and field trials

Answer 29

Aim to harness the genetic diversity of teosinte to increase leaf angle in maize and thus optimise branching architecture

Comparison of maize and teosinte genomes by Tian et al (2019) identified two alleles (UPA1 and UPA2 - both involved in BR biosynthesis) which were determined to be important in determining leaf angle in teosinte and crop maize

UPA1 is an enzyme which catalyses the final step in BR biosynthesis, while UPA2 is a gene which regulates it:
-> Specifically, the UPA2 cis-regulatory element is bound by the negative regulator DRL
-> DRL directly inhibits LG1
-> LG1 induces expression of RAVL1, which in turn promotes UPA1 expression, leading to BR production and wider leaf angles

However, the teosinte UPA2 allele (tUPA2) binds DRL more strongly, leading to increased inhibition of LGL, and leading to reduced UPA1 expression and BR biosynthesis, resulting in smaller auricles and a smaller leaf angle

Therefore, recombinant inbred lines of high-yielding maize varieties and teosinte were created, with almost the entire maize genome, but with the tUPA2 allele introgressed into the genome
-> this increased grain yield at higher planting densities
-> CRISPR was also used as an alternative method to directly edit the genome and induce the same effect (and, again, grain yield at high planting densities was increased)

Question 30

What are the important downstream events following BR signalling that explain how BR actually regulates leaf angle?

Answer 30

Firstly, remember that BIN2 is repressed in the presence of BRs, meaning BES1 is not inhibited, and is free to inhibit CYC4U;1 (a U-type plant cyclin)
-> this prevents CYC4U;1 from forming an active complex with CDKA and promoting cell cycle progression in m2 schlerenchyma cells
-> Therefore, proliferation in the m2 region is inhibited, while BR simultaneously PROMOTES elongation of inner m1 cells
-> Overall, this leads to enlarged lamina inclination, and hence a wider leaf angle

Meanwhile, in the ABSENCE of BRs, BIN2 is active and inhibits BES1, so CYC U4;1 is expressed and forms an active complex with CDKA, leading to proliferation of sclerenchyma cells in the m2 region, resulting in a thickened outer lamina joint, and thus a smaller, more erect leaf angle

Question 31

Given that BRs widen leaf angle (via the pathway described in a previous FC), does this mean that decreasing BR signalling in (for example) rice would improve yield?

Answer 31

Not necessarily - decreased BR leads to more erect leaf angles (which may improve the efficiency of plant architecture for capturing solar radiation) BUT also results in smaller grains, therefore there is no overall increase in grain yield

Ideally, specific manipulations are needed which could induce dwarfism, erect leaves AND big grains simultaneously, with a goal-directed overall design of the plant

As such, the more we research and understand these complex pathways and outputs, the more efficiently and successfully these kind of manipulations can be used and targeted.