Lecture 6a: Abiotic stress: heat stress Flashcards
Key points from previous lectures
*Discussed some aspects of drought and salt stress signalling, highlighting the commonalities between both stresses
*Second messengers, ROS, plant hormones
*Activating kinases – leading to activation of transcription factors, or other functional proteins (i.e ion channels, Na+/H+ antiporters)
*Examples of forward and reverse genetic approaches to identify and engineer different parts in stress perception and signalling for improvement of stress tolerance
*Identification of genes underlying phenotypes - Natural variation, random mutagenesis
*Functional evaluation of candidate genes for stress phenotype – T-DNA insertion, RNAi, CRISPR/Cas9
Questions and key issues
*How much do we understand how (some) plants respond to and/or tolerate stress?
*How useful have forward and reverse genetic approaches in model systems been in identifying the genes behind this?
*If a gene looks promising after preliminary testing, is it easy to introduce it into/ modify it- in crops?
*What additional potential problems could there be when taking lab developed crops to field testing?
*What are the other obstacles to bringing desired product to market?
Temperature increase is sustained by increased
atmospheric concentrations of greenhouse gases
C02 in the atmosphere
see gas pie chart from http://www.ipcc.ch/publications_and_data/ar4/syr/en/spm.html
Carbon dioxide (CO2) - Fossil fuel use is the primary source of CO2.
Important source of CO2, i.e. deforestation (often for grazing land)
Land can also remove CO2 from the atmosphere through reforestation.
Methane (CH4) - Agricultural activities, waste management, and energy
Nitrous oxide (N2O) - Agricultural activities, such as fertilizer use
Fluorinated gases (F-gases) - industrial processes, refrigeration, variety of consumer products - include hydrofluorocarbons (HFCs perfluorocarbons (PFCs), and sulfur hexafluoride (SF6)
Some chemicals have been banned from use and this has resulted in some ozone recovery
Global warming is driving climate change e.g. the recent 2022 heatwave/drought in Europe
Definition of heat stress
*Temperatures hot enough for a sufficient time to cause irreversible damage to plant function or development
*High temperatures increase the rate of reproductive development, i.e. shortens the time for photosynthesis for growth to contribute to fruit or seed production – does not cause irreversible damage but may limit yield
The temperatures where you get irreversible damage vs yield limitation really depends on the plant species and the climate it is adapted to
Crop species and cultivars differ in their sensitivity to high temperatures
The CO2-concentrating system of C4 plants, which compensates for Rubisco inactivation by increasing the CO2 level around Rubisco.
Photorespiration function Rubisco enzyme can out-compete the carboxylation functionality in the absence of a CO2-concentrating mechanism under stress conditions
The time of day also plays a large role in temperature exposure
Night-time heat can particularly impact wheat crop yield – posing a threat to food security
Over night plants are less able to handle stress as this requires energy derived from photosynth. This means that the plant will have to break down storage carbs to respond – thus reducing yield mass
see:
Night-time heat is killing crops. Scientists are
rushing to find resilient plants:
Night temperatures are rising fast and that’s
a problem for rice and other critical crops.
which have fever defences at night
by Olivia Paschal in Jonesboro. Arkansas
link:
https://www.theguardian.com/environment/2022/sep/01/heat-resistant-crops-hotter-night-temperatures-climate
Why is a night-time temperature rise so damaging?
Recent research on this topic: https://www.pnas.org/doi/10.1073/pnas.2025899118
- affect the timing of molecular activities
T at night are rising significantly faster than day temperatures, which is a big problem for humans, animals but also for plants. Plants have fewer defense mechanisms available at night,
Every 1C rise in night-time temperatures could cause wheat yields to drop by 6% and rice yields by as much as 10%. Hotter nights can also affect quality, making the rice chalky and less palatable and can even change its nutritional composition.
Plant responses to high temperatures
*Most tissues of higher plants are unable to survive extended exposure to temperatures above 45°C
*Non-growing or dehydrated tissues (seed, pollen) survive heat better than hydrated tissues, i.e. dry seeds can endure 120°C
see table of tolerance for diff plants and seeds in lecture notes. Source Table 11.2 in Levitt 1980
Maize is a C4 plant and therefore more tolerant to heat but less productive in cold conditions therefore cannot grow in cold climate such as north England
Chloroplast response to heat stress
heat shock:
a. Heat inactivation of PSII
b. Chlorophyll break down
c. Inactivation of Rubisco activase
d. Impairment of protein translation
heat stress:
a. Protective protein chaperones
b. Retrograde signaling pathways
c. Chloroplast Protein Quality Control
link: https://www.frontiersin.org/files/Articles/520328/fpls-11-00375-HTML/image_m/fpls-11-00375-g001.jpg
Heat impacts timeline
Effect of heat on plants step by step:
Loss of membrane integrity - excessive fluidity of membrane lipids at high temperature - loss of physiological function
Photosynthesis is particular sensitive to high
temperatures mainly due to membrane instability
Production of reactive oxygen species (ROS)
Metabolic and cellular disequilibria, leading to cell
death
ROS causes bleaching
Sequential reduction of oxygen through the addition of electrons leads to the formation of a number of ROS including superoxide, hydrogen peroxide, hydroxyl radical, hydroxyl ion
Radicals with at least one unpaired valence electron indicated in red (see diagram in notes)
Hydroxyl radicals are extremely damaging
During a typical summer day
Early morning plants sense and evaluate the need for heat stress defences they may or may not require by mid-day in order to prevent heat damage and repair it by mid evening.
Activity of lipid desaturases at different temperatures may set activation thresholds to distinguish between mild and harsh increases in temperature. Within minutes they can emit a transient heat shock signal to produce HSPs and initiate repair and induce ROS scavenging metabolites. During a hot summer this could occur on a daily basis.
See graph from:
Jianing G, Yuhong G, Yijun G, Rasheed A, Qian Z, Zhiming X, Mahmood A, Shuheng Z, Zhuo Z, Zhuo Z, Xiaoxue W and Jian W (2022) Improvement of heat stress tolerance in soybean (Glycine max L), by using conventional and molecular tools. Front. Plant Sci. 13:993189. doi: 10.3389/fpls.2022.993189
Transcriptional response to heat: top 100 genes
see graph Jianing G et al (2022)
A- RNASeq of heat treated Arabidopsis seedlings – top 100 most heat induced genes. The top genes are exclusively chaperones (red) Other inducible genes are calcium binding genes, metabolic genes and transcriptional regulators (yellow)
B- chaperone abundance in control (green) and heat treated (red) samples – high upregulation in response to heat stress
Potential mechanisms for heat shock regulation
How do plants feel the heat? Trends in Biochemical Sciences, October 2022, Vol. 47, No. 10 https://doi.org/10.1016/j.tibs.2022.05.004
Calmodulins (CaMs): calcium-binding proteins that typically bind other proteins, such as cyclic nucleotide gated channels. (recognise ca influx)
Upon binding of entrant periplasmic Ca+2 ions, CaMs change their conformation and send a specific cellular signal to produce the heat shock response (HSR)
Heat shock proteins (HSPs): proteins that massively accumulate in response to mild HS. Many HSPs are molecular chaperones. Others are heat shock signaling and transcription factors and enzymes that produce thermo- and ROS-protective metabolites. Other HSPs have unknown functions
High leaf temperature and water defecit
Energy input:
sunlight absorbed by leaf
Heat dissipation:
long-wavelength radiation
conduction and convection to cool air
evaporation cooling from water loss
Heat stress direct effect:
associated with hot tissue temp.
indirect effect:
associated with plant water deficit that can arise due to high evaporation demands
Plants may develop thermotolerance
Acquired thermotolerance (AT): ability of a plant to accumulate HSPs and thermo- and ROS-protective metabolites, in response to a mild and harmless, prior warming, conferring the ability to survive an upcoming severe harmful HS for a few hours. A is w/out acclimatisation B is with.
Periodic brief exposure to sub-lethal temperatures induces tolerance to otherwise lethal temperatures (induced or acquired thermotolerance)
Hence, the heat stress response is a genetically controlled process that can be stimulated by mild or sub-lethal temperatures and further trigger the onset of heat stress response in. The heat-stress response in plants is mainly conserved via cellular compartments and regulatory networks some of which we will explore in more detail
Transcriptional Regulatory Network of Plant Heat Stress
Key players are HEAT SHOCK TRANSCRIPTION FACTORS (HSFs)
The HSF family is conserved across nearly all life forms
Plants have the most HSFs – we do not know the function of all of those present in A. thaliana etc.
See reviews: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3550655/pdf/12298_2008_Article_14.pdf
https://www.frontiersin.org/articles/10.3389/fpls.2023.1111875/full
^ good overview of strategies including pathway cross talks
& see diagram in notes from:
Ohama et al 2016 Trends in Plant Science https://doi.org/10.1016/j.tplants.2016.08.015
During the HSR, HEAT SHOCK TRANSCRIPTION FACTOR A1s (HsfA1s) act as the master regulators of transcriptional regulation. Under nonstress conditions, HEAT SHOCK PROTEIN 70 (HSP70) and HSP90 repress the activity of HsfA1s. Upon heat shock, HsfA1s are activated and induce the expression of transcription factors, such as DEHYDRATION-RESPONSIVE ELEMENT BINDING 2A (DREB2A), MULTIPROTEIN BRIDGING FACTOR 1C (MBF1C), HsfA7s, HsfA2, and HsfBs to upregulate or fine-tune the expression of heat stress (HS)-inducible genes. In addition, JUNGBRUNNEN1 (JUB1), NAC019, DREB2C, and other HsfAs are involved in the HSR and constitute a complex regulatory network. In the endoplasmic reticulum (ER), bZIP28 acts as an important transcription factor for coping with ER damage independent of HsfA1s. Post-translational regulation is necessary to stabilize and activate DREB2A. Activated DREB2A induces the expression of its target genes, including HsfA3, through a trimer comprising DNA POLYMERASE II SUBUNIT B3-1 (DPB3-1), NUCLEAR FACTOR Y, SUBUNIT A2 (NF-YA2), and NF-YB3. Histone modification and miRNAs are emerging as factors involved in transcriptional regulation and HS memory. Transposon expression is activated during the HSR, although DECREASED DNA METHYLATION 1 (DDM1) and MORPHEUS MOLECULE 1 (MOM1) quickly reset their expression. However, ONSEN is highly activated by HsfAs and shows potential for transposition.
HSFs regulate other stress responses
Heat shock transcription factor (HSF) genes have been implicated not only in
thermotolerance but also in plant growth and development, and therefore could
influence water productivity.
HSFA1b over-expression improves both basal and acquired thermotolerance. The knockout mutant in two of the 4 HSFA1 genes is sensitive to heat stress.
See:
Arabidopsis HEAT SHOCK TRANSCRIPTION FACTOR A1b overexpression enhances water productivity, resistance to drought, and infection
At:
Bechtold, U., Albihlal, W.S., Lawson, T., Fryer, M.J., Sparrow, P.A.C., Richard, F., Persad, R., Bowden, L., Hickman, R., Martin, C., Beynon, J.L., Buchanan-Wollaston, V., Baker, N.R., Morison, J.I.L., Schoffl, F., Ott, S. and Mullineaux, P.M. (2013) ‘Arabidopsis HEAT SHOCK TRANSCRIPTION FACTOR A1b overexpression enhances water productivity, resistance to drought, and infection’, Journal of Experimental Botany, [online] 64(11), pp. 3467–3481. Available at: https://doi.org/10.1093/jxb/ert185
HSFA1b OE-Abiotic stress tolerance
The mutant plants are also more drought tolerant and show improved water productivity (“more crop per drop”). More seed yield per plant per ml water used.
Plants were grown in identical amount of soil and were watered to a target weight every day and the amount of water was monitored every day.
^ (This can now be done using automated systems, we did that by hand.)
You can calculate life time water use over the entire experiment, and get biomass data (above ground) once the plant is harvested. Very time consuming and labour intensive experiment. 100 plants means around 3-4 hours or watering per day every day!
HSFA1 - class TFs functional characterisation
how do they do it?
* Increased tolerance to heat
*Resistance to virulent Pseudomonas syringae DC3000
*Resistance to oomycete Hyaloperonospora
arabidopsidis pv. WAC09
*Influences acclimation to high light
* Increased tolerance to dehydration
* Regulation of growth and development
*Resistance to Botrytis cinerea
Experimental strategy for HSFA1
Identify promoter regions that are bound by HSFA1b under control and heat stress conditions – these are the direct targets
Compare this with genes that are differentially expressed under control and heat stress conditions.
We can built a picture of genes that are DEG and directly targeted by HSFA1b, and genes that are DE but are indirect targets. This allows us to build a hierarchical network
Read paper Albihlal et al 2018 Bot 69:2487-28632 for understanding
Venn diagram showing the overlap between HSFA1b target genes scored from the ChIP-seq data (Supplementary Data S1) and the target genes bound by HSFA1b (HSF3) from the Arabidopsis Cistrome Atlas
HSFA1b binding changes under heat stress
Three histograms showing the frequency of HSFA1b binding relative to the distance from the TSS of target genomic features in Groups I–III. (E) Pie charts showing the distribution of HSFA1b binding on target genomic features in Groups I–III.
HSFA1b directly regulates TF gene expression
potentially controlling indirectly the expression of
more genes
HSFA1b is a master regulator of >100 other TFs
HSFA1b can indirectly control expression through its regulation of TF gene expression. (A) Heat map comparing normalized FPKM values for 28 TF genes bound by HSFA1b and differentially expressed in 35S:HSFA1b and WT HS plants. Arrows indicate development-associated TF genes.
TF binding data from the cistrome database
We can use public databases to identify the targets of the HSFA1b bound transcription factors.
If these targets are differentially expressed in HSFA1b over-expressing plants we can further built the hierarchical network. HSFA1b –> TF -> a subset of differentially expressed genes. Different branches may then be responsible for different responses we see in the HSFA1b over-expressing plants.
An overview of a Cytoscape-generated HSFA1b hierarchical TF gene network using the data outputs from the Cistrome Atlas with the ChIP-seq and RNA-seq data from this study. The yellow node is HSFA1b, red nodes are TF genes bound by HSFA1b, and blue nodes are differentially expressed TF genes that respond to HS and HSFA1b overexpression, are not bound by HSFA1b, but are scored as binding to the red node TFs.
Venn diagram showing the overlap between HSFA1b target genes scored from the ChIP-seq data (Supplementary Data S1) and the target genes bound by HSFA1b (HSF3) from the Arabidopsis Cistrome Atlas
Albihlal et al 2018 see prev slides for ref
