Lecture 6b: Abiotic stress: cold and freezing Flashcards
Low temperature stress tolerance
Plants can be classified as either chilling-sensitive or chilling-tolerant.
Chilling-sensitive plants e.g. tropical plants are damaged by low positive temperatures, typically around 10-15oC.
Most temperate plants are chilling-tolerant. Such chilling-tolerant plants can be subdivided into plants which can cold acclimate and those which cannot.
Most temperate plants can increase their freezing tolerance through the process of cold acclimation.
Cold climate crops such as wheat and barley have some constitutive freezing tolerance but can gain further freezing tolerance through cold acclimation
How big a problem is freezing?
crop failure due to freezing in 2011:
January : India -Suffered the coldest winter in 30years damaging crops of wheat, soybean, peas, opium and oranges.
February : Mexico –90% of corn and other crops damaged or destroyed by freezing temperatures causing food prices to triple. Tomatoes usually costing $6.95 increased to $22.95.
July 2011: Zimbabwe –Frost destroyed crops of maize, tomatoes, cabbages and flowers for export.
Other issues unseasonal frosts – see: https://www.nature.com/articles/s41558-021-01090-x.pdf
The problems caused by freezing stress
Freezing causes production of ROS which goes onto have other secondary effects (discussed later), desiccation and protein dysfunction ( too cold for proteins to fold and operate properly). Desiccation leads to several different forms of membrane damage. Which, partly depends on the nature and severity of the freezing. Expansion-induced-lysis, lamellar-to hexagonal- II phase transitions, and fracture jump lesions are all forms of membrane damage. Thus, a key function of cold acclimation is to stabilize membranes against freezing injury. In addition, freeze-induced membrane damage results primarily from the severe dehydration associated with freezing. Fracture jump lesions are another form f membrane breakage that is associated with altered membrane structure and also with cellular membranes “sticking” to one another and causing breakages. Expansion-induced lysis occurs when cell volume reduces after water leaves the cell in response to extracellular ice formation and then upon thawing, rushes back in whilst the cell volume is still too small to accommodate it and membranes around the cytoplasm consequently “pop”.
Freezing stress process in cells
Similarities to desiccation stress as many of the cell’s freezing tolerance mechanisms are the ones used in combatting desiccation.
As temperatures drop below 0°C, ice formation is initiated in the intercellular spaces due to the extracellular fluid having a higher freezing point (lower solute concentration) than the intracellular fluid. So the first place water freezes is in the cell wall. This leads to water being drawn out of the cell cytosol (symplast) into the cell wall (apoplast) due to the enormous water potential gradient. Because the chemical potential of ice is less than that of liquid water at a given temperature, the formation of extracellular ice results in a drop in water potential outside the cell. (No “liquid water” outside and lots inside.)
Consequently, there is movement of unfrozen water down the chemical potential gradient from inside the cell to the intercellular spaces.
Types of freeze/thaw response
Expansion-induced lysis (EIL) with endocytotic vesiculation
Freeze dehydration + Contraction
Thawing + expansion
Loss of osmotic responsiveness with endocytotic vesiculation
Freeze dehydration + contraction
Thawing + No expansion
Loss of osmotic responsiveness with exocytotic extrusions and the fracture-jump lesion
Freeze dehydration +contraction
Thawing + No expansion
Freezing temperatures favour formation of non-bilayer structures that disrupt membrane function
Lamellar to hexagonal-ll phase transition
membrane rearrangement resulting in loss of embedded protein function and longterm damage
Cold acclimatisation: solving the problems caused by freezing stress
For arabidopsis three days (or more) at low positive temperatures means the plant can later survive freezing this is cold acclimitisation.
During cold acclimation there is a huge amount of transcriptional reprogramming happening. Thousands of genes change in their expression- not all of them increase, a sizeable proportion decrease.
174 TFs are amongst these. Many changes in gene expression in freezing tolerance aquisition
Transcription factors are involved in switching on cold acclimatisation (CA) genes
Cold and freezing signalling overview
Fig. 1. Schematic illustration of the putative mechanism of cold response regulation in a cell by the cold-responsive transcription factors. The cold stress under freezing temperature is sensed by the membrane receptors on the cell membrane modulating the membrane fluidity for the signal transduction. The signals from the membranes induce cAMP, Ca2+ and reactive oxygen species (ROS) mediated signal transduction via Calcineurin-B Like proteins (CBL), CPKs, CIPKs, CDPKs to the nucleus through a pathway mediated by ICE-CBF/DREB transcription factors. Here, the WRKY, bZIP, MYB, CBF/DREB and NAC transcription factors regulate gene expression for the activation of stress-responsive genes i.e. CORs. The chloroplast under freezing temperature, modulate its PSII light extraction complex and signals for the cold stress response to the nucleus via ROS production. The final product of the COR genes result in physiological responses against cold stress.
Calcium signalling to trigger cold response
Schematic of the signaling network in response to low temperature stress
(see notes)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5684653/
Schematic representation of signaling network in response to low temperature stress. It shows the early events of cold perception, leading expression of COR genes regulated by CBF transcription factors. Characterized Ca+2 sensor proteins and their target proteins are also shown. Refer to text for detailed descriptions. CBF/DREB, C-repeat binding factors/DRE-binding proteins; CBL/CIPK, calcineurin B-like protein/CBL-interacting protein kinase; CAMTA, Calmodulin-binding transcription activator; COR, cold-regulated; HOS, high expression of osmotically responsive gene; ICE1, inducer of CBF expression 1; RCI1A, Rare cold inducible 1A and ADH1, Alcohol dehydrogenase 1; ABF1, ABRE binding factor 1; EIN3, Ethylene Insensitive; HOS1, high expression of osmotically responsive genes; SIZ1, E3 SUMO ligase.
Many cold gene promotors contain the CRT/DRE motif
Many cold up regulated genes contain a consensus sequence, a CCGAC motif in their promoters (“C-repeat” or CRT).
. Thomashow’s lab found the transcription factors that bind this (1997)
. Shinozaki group published in 1998 and called them DREB1 family - previously identified DREB2 - drought responsive TFs
Mike Thomashow’s group (late 90’s)used Y1H assays to find which TF binds this motif. See Stockinger et al 1997 Proc Natl Acad Sci 94 1035.
They called the protein that bound C-repeat binding factors and they demonstrated they had transcriptional activating ability.
Gilmour et al 2004 in Plant Molecular biology (same lab) later showed CBF2 and CBF3 are similar. http://www.ncbi.nlm.nih.gov/pubmed/15356394
Shinozaki group published a year later and called them DREB1 family. (This is the group that named the drought responsive transcription factor DRERB2 that we saw earlier. Liu et al 1998, The Plant Cell)
Remember the CRT/DRE motif from the drought response pathway?
low temp response is highly similar to heat induced osmotic stress
Well-studied classes of TF that respond to osmotic stress are the AREB/ABF group and the DREB2 group. The DREB2A gene was isolated as a gene encoding a DRE/CRT-binding protein and was shown to be induced by osmotic stresses.
A very simplified diagram showing the roles of the DREB and ABF transcription factors and their cis elements in cold and drought gene activation. There are many review articles showing a much more detailed picture, which is being added to all the time. E.g. see From a review article: Nakashima, K., Ito, Y. and Yamaguchi-Shinozaki, K. (2009). Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiol. 149: 88-95.
This article shows diagrams for rice and Arabidopsis mostly based on research from the Shinozaki group in Japan.
CBF1-3 are Rapidly Induced in Response to Low Temperature
TFs upregulated resulting in target gene activation response
Gilmour et al (1998) Plant J. Vol 16; 433-. https://onlinelibrary.wiley.com/doi/full/10.1046/j.1365-313x.1998.00310.x
Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression.
CBF1-3 are arranged in a tandem repeat in the genome – gene duplications
(Also from the Thomashow group). Shows expression of the three CBF genes is cold- inducible and precedes the expression of presumed target genes but doesn’t show causality
CBF genes encode AP2/ERF (APETALA2/Ethylene-Responsive Factor)-type transcription factors that specifically bind to the C-repeat (CRT)/dehydration-responsive element (DRE; G/ACCGAC) and regulate the expression of downstream cold-responsive (COR) genes
CBF Genes Act as “Master Switches” to Activate the Cold Acclimation Response
Jaglo-Ottosen et al (1998) Science. Proof that CBFs switch on many target COR genes and thus act as a master switch for cold acclimation. Plants behave as if cold acclimated even if they have never seen the cold.
This works if any of the three CBF genes are overexpressed.
You will also see in the literature this is also used to protect plants from drought as they are the same genes as switched on by the DREB2 proteins (see pathway on previous lecture slides).
CBF genes encode AP2/ERF (APETALA2/Ethylene-Responsive Factor)-type transcription factors that specifically bind to the C-repeat (CRT)/dehydration-responsive element (DRE; G/ACCGAC) and regulate the expression of downstream cold-responsive (COR) genes
Loss of function of all 3 CBFs show that all 3 are required for CA and freezing tol.
https://pubmed.ncbi.nlm.nih.gov/27252305/
Only in 2016 was
Figure 2. cbf triple mutants are very sensitive to freezing. A, Electrolyte leakage assay conducted on nonacclimated (NA) Col-0, cbf123-1, and cbf123-2 plants grown at 23˚C. B, Electrolyte leakage assay performed on Col-0, cbf123-1, and cbf123-2 plants after cold acclimation (CA; 4˚C for 7 d). C and D, Freezing survival assay
If you knock out all CBF genes plants cannot tol cold confirming the role of CBFs
CBFs exist and are active in crop species: see homologues in canola, rye and wheat
Jaglo et al (2001) Plant Physiology 217: 910-7 Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species http://www.plantphysiol.org/content/plantphysiol/127/3/910.full.pdf
Accumulation of CBF and putative target gene transcripts in response to low temperature. Plants were grown at normal growth temperatures (20°C–22°C) and transferred to low temperature (4°C) for the indicated times. Total RNA was isolated from leaves and northern analyses performed using probes for CBF transcripts and putative CBF-targeted cold-regulated genes for B. napus (Bn115), wheat and rye (Wcs120/COR39), and Arabidopsis (COR15a) as described in “Materials and Methods.” At, Arabidopsis; Bn, B. napus; Sc, rye; Ta, wheat.
Shows that CBF like genes occur in crops and are inducible by cold. Also shows they are induced prior to the induction of known crop cold genes however doesn’t prove they are responsible. See later for the proof.
Perfect targets for improving crop freezing tolerance?
However, …. Constitutive Expression of CBF is Detrimental to Plant Growth
Plants “think” it is cold and adjust their
physiology and biochemistry in a way
that is not optimal for growth and development at warm temperatures
CBF1 increases growth repression by increasing amount of DELLAs
CBF1 Represses Growth and Delays Floral Transition through the GA-Signaling Pathway
CBFs promote transcription of the genes for RGL3 and GA oxidases and this is what leads to dwarfing. This was discovered by Achard et al 2008 https://www.ncbi.nlm.nih.gov/pubmed/18757556
Constitutive expression of CBF1 confers freezing tolerance but also slows growth
Low temp response inhibits giberellin resulting in reduced growth
^spraying plants with gibberelic acid allows the CBF mutants to regain normal growth pattern
HvCBF2 increases FT but causes stunting in barley
Freeze-tol spring barley genetically edited – tol results in reduced growth
Constitutive expression of CBFs is therefore not beneficial – inducibility is needed
Hv-CBF2A overexpression in barley accelerates COR gene transcript accumulation and acquisition of freezing tolerance during cold acclimation
In Plant Molecular Biology (2013) Zoran Jeknić et al. Available at http://link.springer.com/article/10.1007%2Fs11103-013-0119-z
DT (Dicktoo) is a winter hardy variety of barley. The CBF2 gene was taken from DT and the less hardy spring barley gold promise (GP) transformed with a construct to express it constitutively.
Study of potato with constitutive vs inducible CBF expression
inducible stress response for freeze tol that will not inhibit growth outside of these conditions
Pino et al (2007) in Plant Biotechnology Journal
Volume 5, Issue 5, pages 591-604, 8 JUN 2007 DOI: 10.1111/j.1467-7652.2007.00269.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1467-7652.2007.00269.x/full#f3
AtCBF1 and AtCBF3 overexpression via the 35S promoter increased freezing tolerance about 2 °C, whereas AtCBF2 over-expression failed to increase freezing tolerance. Transgenic plants of AtCBF1 and AtCBF3 driven by the rd29A promoter
Effect of AtCBF1‐3 genes controlled by constitutive or cold‐inducible promoters on growth characteristics of Solanum tuberosum plant phenotype. A representative 16‐week‐old plant growing at 25 °C is shown for each indicated line. Panels are sized to the same approximate scale. WT, wild‐type.
35S is a constitutive promoter. Rd29a is a cold and drought inducible promoter.
Stress-inducible promoter for CBF increases freezing tolerance
Freezing tolerance time course of rd29A::AtCBF3 lines. Samples were collected and analysed at the indicated days over a 2‐week period at 2 °C. The 0 h control was collected from respective plants growing at 25 °C as a warm control. LT50 (–°C) values of leaf tissue samples of wild‐type (WT) and three rd29A::AtCBF3 transgenic lines were determined as in Figure 2.
https://onlinelibrary.wiley.com/doi/full/10.1111/j.1467-7652.2007.00269.x
ROS production and detoxification
Identifying more subtle areas of freeze tol. - ROS breakdown enzyme activation
Figure 9.4 from Plant Biotechnology, Slater et al. (course text book).The combination of oxygen with an unpaired electron starts a chain of reactions that reduce damaging ROS. However, enzymes exist that can detoxify these damaging chemical species. Many of these have been tested as means of improving crop stress tolerance.
Antioxidants in transgenic plants
A very brief summary of the contents of Table 3 in Gill and Tuteja’s 2010 review. https://www.ncbi.nlm.nih.gov/pubmed/20870416
Table 3. ROS scavenging enzymatic and non-enzymatic antioxidants and their role in transgenic plants for abiotic stress tolerance
Ontario field trial: alfalfa overexpressing Superoxide Dismutase
McKersie, B.D., Bowley, S.R. & Jones, K.S. Winter survival of transgenic alfalfa overexpressing
superoxide dismutase. Plant Physiol. 119, 839–848 (1999).
Chart re-drawn from the date in Table VII of the paper
No survival in control – elevated ROS breakdown enzyme expression in mutants increased survival rate
Compatible solutes Glycine betaine accumulation
. Ammonium compound that occurs naturally in plants, animals and microorganisms
. It accumulates in some plant species at elevated levels in response to different abiotic stresses
. Has been used to improve abiotic
stress tolerance, of which there are
many examples in literature
See Image in notes from: https://www.mdpi.com/2073-4395/12/2/276
Other examples: https://www.frontiersin.org/articles/10.3389/fpls.2018.01995/full
Glycine betaine naturally produced in plant cells improves freeze tol
Glycine betaine in freezing tolerance in mangroves
. Glycine betaine accumulation was similar across all source populations and
freeze exposure locations
. Glycine betaine accumulation does not limit range adaptation but is used for
freezing tolerance irrespective of where populations come from
https://besjournals.onlinelibrary.wiley.com/doi/full/10.1111/1365-2745.13243
Observed accumulation of beataine in mangrove tissue allowed survival in freezing temp. But was not increased in populations more often exposed to freezing conditions
see:
Hayes, M.A., Shor, A.C., Jesse, A., Miller, C., Kennedy, J.P. and Feller, I. (2019) ‘The role of glycine betaine in range expansions: protecting mangroves against extreme freeze events’, Journal of Ecology, 107(6), pp. 2657–2668. Available at: https://doi.org/10.1111/1365-2745.13243
