4.11 lecture 2: wood from the trees Flashcards
Photosynthesis captures light energy as reduced carbon
6 CO2 + 6 H2O > C6H1206 + 6 O2
(CO2+H2O >rubisco> O2 + sugars)
from :Energy input from sunlight
&”Low energy” oxidized carbon in carbon dioxide
to: Oxygen is released as a by-product
& “High energy” reduced carbon
1/7th of atmospheric carbon is fixed each year
Almost all organic carbon is fixed into organic form by Rubisco
photosynthesis combines carbon fixing and light dependent reactions
Light-dependent reactions: The first step is the conversion
of light energy into ATP and reducing power, NADPH
Carbon fixation reactions: The power from ATP and NADPH is used to make high-energy sugars from CO2
see diagram in notes from: Buchanan, B.B., Gruissem, W. and Jones, R.L. (2000) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists
Cellulose is the most abundant biopolymer on earth
see diagram from PNAS June 19, 2018 115 (25) 6506-6511; https://doi.org/10.1073/pnas.1711842115
plants turn atmospheric carbon into renewable resources
Trees are a renewable source of biomaterial & in the immediate term, forestry residues are a source of energy in biomass generators
current biomass as energy source is palm oil and american wood chips – not sustainable
Process from wood to biomass energy generation:
- feedstock, harvest and logistics
- pre-treatment
- enzymatic saccharification
- fermentation and product formulation
^ See: Journal of Industrial Microbiology & Biotechnology volume 47, p623–657 (2020)
Bio-ethanol production also results in greenhouse gas pollution
Farrell et al 2006 Science 311, p506
it is important not to use food crops for biofuel generation
Plant vascular tissue is highly ordered
Plant vascular tissue is highly ordered intrinsically
Nutrients travel in the phloem and water in the xylem, these tissues are separated by the cambium
Arabidopsis has long been used as a model organism for plant (genome) study due to:
- Small genome
- Small size
- Obligate self-pollinator
- Easy to transform
- Mutants available for most genes
PXY signalling regulates the vascular cambium
PXY = phloem intercalated with xylem
^ named after the mutant phenotype.
In this mutant it becomes hard to define where these three tissues occur – potentially a change in orientation of cell division?
How PXY functions
PXY encodes a receptor like kinase – sits in the plasma membrane and receives signals,
when it receives a signal it sends it by phospho-relay to the nucleus to change gene expression
TDIF/CLE41 – trans differentiation inhibiting factor is the ligand that binds PXY
(CLE41 is the gene that is translated and cleaved down to the active peptide TDIF)
PXY has 4 signalling outputs
- Vascular proliferation:
Stimulating PXY increases vascular cell division by activating WOX4 and WOX14 which stimulate proliferation.
We know this role of WOX4 and WOX14 because if they are mutated they lose their function and cell proliferation reduces - Represses xylem differentiation by degrading BES1(gene which promotes xylem differentiation)
- Vascular organisation - activates LBD4 expression (gene controlling vascular bundle shape) to specify boundary shape.
LBD4 is required for TDIF overexpression phenotypes
Gene expression in space and time is really important in cell biology, adjustments can result in deformity and loss of functions
CLE41 in the wrong place results in expression of cell types in the wrong quantity,alignment and location
- Cambium initiation - PXY is required but its function in cambium initiation is unknown at present
True radial growth occurs a few days after germination
see: high levels of auxin signalling define the stem cell organiser of the vascular cambium Smetana et al.
PXY is crucial for promoting the stem cells to divide
PLT3 and PLT5 are putative PXY targets (identified by RNAseq)
PLETHORA (PLT) genes were previously shown to be required for lateral root initiation
PLT expression shown to be induced by PXY ligand - detected by fluorophore tagging
PLT3/5 necessary for ectopic cambium division
Do PLT and PXY combine to initiate cambium formation?
research outcomes:
PLT3 and PLT5 demonstrate increased expression when PXY signalling is increased.
PLT3 and PLT5 demonstrate reduced expression when PXY signalling is lost.
PLT genes are rapidly induced upon TDIF application.
PLT genes are required for ectopic cell divisions present in 35S::CLE41 lines
conclusion:
· PLT3 and PLT5 expression is promoted by PXY signalling
. PLT genes are required for PXY- regulated cell division
. PLT genes are required for timely cambium initiation
Increasing tree productivity:
Can manipulating PXY signalling increase tree productivity?
Simple overexpression didn’t work:
Constitutive over-expression of CLE41 led to:
(i)Increases in cambium cells (good)
(ii)Reductions in xylem differentiation (bad)
(iii)Failure to organise vascular tissue (bad)
(iv)Ectopic cambium initiation (bad)
Therefore to benefit from increase in cambium cells a transgenic overexpression line needed to be generated
Crown gall disease: the tumour inducing principle
The first written record of crown gall disease, on
grape, dates from 1853
Fridiano Cavara (1897) found that a bacterium causes crown gall in grape
Crown gall induces growths at wound sites and severely limits
crop yields and growth vigor
Crown gall induction:
The disease is caused by Agrobacterium tumefaciens.
Agrobacterium transfers a T-DNA to plant cells: Tumor-inducing genes and opine synthesis genes
Agrobacterium hijacks plant cells to make opine, a nutrient source for the bacteria. Opines induce expression of genes required for opine uptake and catabolism
The discovery that T-DNA was inserted into the plant genome raised the possibility that any gene could be transferred into plants
The discovery that T-DNA was inserted into the plant genome raised the possibility that any gene could be transferred into plants
T-DNA and the vir genes can be located on separate plasmids or replicons, making cloning easier
Because the Ti plasmid is so large, a binary system was developed to allow gene cloning into a smaller plasmid
The smaller plasmid is introduced into Agrobacterium carrying a helper plasmid with the vir genes
see: Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A. (1983). A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature. 303: 179-180.
Arabidopsis floral dip transformation using the cauliflower mosaic virus
Arabidopsis can be transformed by floral dip:
Replace T-DNA with gene of interest, which is introduced into Agrobacterium carrying vir genes in the binary plasmid
Inoculate plant with engineered Agrobacterium
A process that takes 6 months in trees will take 6 weeks in Arabidopsis
The floral dip method aims to expose cells that will form the embryo (seed) to Agrobacterium. If the agrobacterium is in the embryo it should cause an outcome in the seeds.
To be expressed in a plant cell, a gene has to look like a plant gene with promotor and UTR’s at each end
The cauliflower mosaic virus has a strong, constitutive promoter (35S) to replicate in plant cells - It is not cell type specific.
^ this is an issue as we only want overexpression in the cambium
Ligands can regulate expression of receptor PXY.
When PXY is relieved from negative feedback regulation increased vascular cell division occurs
result: cambium cell increase without neg. effects of simple overexpression - but how to apply to trees?
How could this process identified in Arabidopsis be used to increase tree productivity to converts CO2 into biomaterials at scale?
(i) Identify a forest tree species to transfer this technology to.
(ii) Determine if PXY and CLE41-like genes exist in that species
(iii) If they exist, determine if they perform a similar function as their Arabidopsis homologues
(iv) Manipulate their expression in space and time to increase cell division specifically in the cambium
US Department of Energy invested significant funds into bioenergy research in 2006 - Aka the ‘billion-ton vision’
(i) Identify a forest tree species to transfer this technology to: Poplar
Poplar appeared suitable
see: Nilsson, O., Alden, T., Sitbon, F., Little, C.H.A., Chalupa, V., Sandberg, G. and Olsson, O. (1992) ‘Spatial pattern of cauliflower mosaic virus 35S promoter-luciferase expression in transgenic hybrid aspen trees monitored by enzymatic assay and non-destructive imaging’, Transgenic Research, [online] 1, pp. 209–220.
Determine if PXY and CLE41-like genes exist in that species: PXY and CLE41 homologues are present in poplar
PXY homologues in Poplar
see: WUSCHEL-RELATED HOMEOBOX4 (WOX4)-like genes
regulate cambial cell division activity and secondary growth in
Populus trees. Melis Kucukogl et al
(iii) Determine if these homologues perform a similar function as their Arabidopsis homologues: yes they do!
See Current Biology 25(8) 1050-1055
In Arabidopsis: PtCLE41 behaves as AtCLE41. PtPXY behaves as AtPXY.
Do these genes control wood formation in poplar? yes
(iii continued)
Agrobacterium mediated transformation of leaf tissue:
Propagation of the plant
1 Cuttings are brought into sterile culture.
2 Soak in bleach
(trade-off- between death of microbes and death of plant).
3 Or surface sterilise seed.
Explant preparation
Pieces of leaf are pre-treated on callus induction media
Co-Culture
Leaf pieces are co-cultured with Agrobacterium to enable T-
DNA transfer
Callus formation
. Co-cultured tissue treated with Antibiotics for 3-4 weeks
(kill Agrobacterium)
. Move to media without antibiotics for 2-3 days
Shoot formation
· High cytokinin in media promotes callus and shoot formation
Transfer to rooting medium
in 2-3 weeks transgenic plantlets form
Finally you have a transgenic plant to transfer to soil
Yes these genes control wood formation in poplar!
Constitutive over expression shows that PtPXY and PtCLE41 regulate wood formation in poplar
From a biotechnology perspective, these lines are useless
Can we manipulate the system to get extra cell divisions that lead to improved wood formation?
If we increase TDIF levels in the phloem, will vascular organisation be maintained?
(iv) Manipulate their expression in space and time to increase cell division specifically in the cambium
The SUC2 gene has a promoter that drives expression specifically in companion cells
Increasing expression of CLE41 in the phloem, increases xylem formation
“Plant growth is the result of cell proliferation in meristems, which requires a careful balance between the formation of new
tissue and the maintenance of a set of undifferentiated stem cells. Recent studies have provided important information on
several genetic networks responsible for stem cell maintenance and regulation of cell differentiation in the apical meristems”
see: Schrader, J., Nilsson, J., Mellerowicz, E., Berglund, A., Nilsson, P., Hertzberg, M. and Sandberg, G. (2004) ‘A high-resolution transcript profile across the wood-forming meristem of poplar identifies potential regulators of cambial stem cell identity’, The Plant Cell, [online] 16(9), pp. 2278–2292
