Lecture 5a: Abiotic stress: drought Flashcards

Question

Natural variation in drought resistance – forward genetics

Answer 1

In the paper below 136 genotypes were screened under drought stress and measured a range of physiological traits *Insight into drought-tolerant genotypes *Best traits to evaluate drought responses *Underlying SNPs with potential candidate genes *May be useful for soybean genetic breeding

Answer 2

Identification of soybean drought-tolerant genotypes and loci correlated with agronomic traits contributes new candidate genes for breeding Key message Drought tolerance level of 136 soybean genotypes, the correlations among traits were evaluated, and sev- eral important drought-tolerant genotypes, traits, SNPs and genes were possibly useful for soybean genetic breeding. Chen, L., Fang, Y., Li, X., Zeng, K., Chen, H., Zhang, H., Yang, H., Cao, D., Hao, Q., Yuan, S., Zhang, C., Guo, W., Chen, S., Yang, Z., Shan, Z., Zhang, X., Qiu, D., Zhan, Y. and Zhou, X-A. (2020) 'Identification of soybean drought-tolerant genotypes and loci correlated with agronomic traits contributes new candidate genes for breeding', Plant Molecular Biology, 102, pp. 109–122. Available at: https://doi.org/10.1007/s11103-019-00934-7

Answer 3

Hundreds of studies have revealed 1000s of genes involved in drought responses Some are regulated by ABA, others are ABA-independent Many different transcription factors are involved Many of the targets are involved in cellular protection or conserving water see diagram in notes from Fukao, T. and Xiong, L. (2013). Genetic mechanisms conferring adaptation to submergence and drought in rice: simple or complex? Curr. Opin. Plant Biol. 16: 196-204

Answer 4

1.Target regulatory genes that effect a large number of proteins e.g.: *encoding transcription factors, *production of signalling molecules *and other processes 2.Do a rapid survey in a model species in the lab (usually Arabidopsis). If positive… 3.Go to field with the same gene or its homolog in crops ^sometimes works sometimes doesn't as crops are diff to arabidopsis

Answer 5

see diagram in notes ^ This article shows diagrams for rice and Arabidopsis mostly based on research from the Shinozaki group in Japan. A very simplified diagram showing the roles of the DREB (Drought gene expression) and ABF transcription factors and their cis elements in drought gene activation. 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. 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.

Answer 6

see diagram in notes Liu Q et al. Plant Cell 1998;10:1391-1406 figure 11 The 6 lines all refer to different treatments. Just look at the first two: control plants and plants allowed to dry out. In wild type (WT) plants (not transgenic) the DREB2 transcript appears in response to drying out. As a result, there is an increase in the transcript level of target genes such as rd29A. The 35S overexpresser lines (a and b are both similar lines) show constitutive expression of DREB2A as expected however, they don’t show the expected increase in rd29A . Overexpressing DREB2 doesn’t change expression of target (RD29A). So perhaps expressing more transcript and more protein isn’t enough. Something needs to happen to the protein to make it work. Liu Q et al. Plant Cell 1998;10:1391-1406 See figure 11 (described below) Expression Analyses of the DREB1A, DREB2A, and rd29A Genes in Transgenic Plants. RNA gel blotting was conducted to measure the amount of DREB1A, DREB2A, or rd29A mRNA in transgenic Arabidopsis plants carrying the 35S:DREB1A transgene, those carrying the 35S:DREB2A transgene (35S:DREB2Aa and 35S:DREB2Ab), and those carrying pBI121 (wt). Transgenic plants were dehydrated (Dry), transferred from agar plates for hydroponic growth in water (H2O), transferred from agar plates for hydroponic growth in 250 mM NaCl (NaCl), transferred from agar plates for hydroponic growth in 100 μM ABA (ABA), transferred to 4°C (Cold), and then treated for 5 hr under each condition or were untreated (Control). DNA fragments for the DREB1A and DREB2A cDNAs or the 3′ flanking region of rd29A were used as probes.

Answer 7

see diagram in notes ^Work of Sakuma et al The Plant Cell 2006: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1456870/ Domain analysis of DREB2A revealed that the central region of DREB2A contains a negative regulatory domain that causes the protein to be degraded by the 26S proteasome under non-stress conditions. So it is not stable for long enough to activate target gene expression. (later work discovered the proteins that make this happen (DRIP1 and DRIP2 = RING E3 ligases) Reference: Qin et al. Plant cell 2008 vol 20 :1693-1707. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2483357/ Deletion of the NRD region makes DREB2A constitutively active (DREB2A-CA) as it does not get degraded. Overexpression of DREB2A-CA in transgenic Arabidopsis induced expected phenotypic changes, (including dwarfism, unfortunately), up-regulation of many stress-inducible downstream genes, and drought tolerance. Similar story for the ABA responsive transcription factor AREB1: it also has a negative regulatory domain. See Fujita, Y., Fujita, M., Satoh, R., et al. (2005) Plant Cell, 17, 3470-3488

Answer 8

see photos in notes ^This works, but the plants are rather dwarfed – a penalty associated with being drought tolerant Sakuma, Y., Maruyama, K., et al. (2006) Plant Cell, 18, 1292-1309 (figure 6) This figure from the same paper mentions DREB1A too. It can also activate drought tolerance as it activates the same CRT/DRE-containing genes. However it doesn’t have the problem of being degraded. Drought and Freezing Tolerance of the 35S:DREB2A CA and 35S:DREB1A Plants. The stress treatments were conducted as described in the text. Drought: water withheld from plants for 2 weeks; freezing: 4-week-old plants exposed to –6°C for 30 h and returned to 22°C for 5 d. (A) Photographs of plants before and after stress treatments. (B) Survival rates of plants exposed to drought and freezing stress. Average survival rates and standard errors were calculated using results of three replicated experiments. Twenty plants (five plants/pot) were tested in each experiment. In all experiments, the plants with asterisks had significantly higher survival rates than wild-type plants ( 2 test, *P < 0.05, **P < 0.01).

Answer 9

Using a CA form of DREB TF Kumar, S., Muthuvel, J., Sadhukhan, A., Kobayashi, Y., Koyama, H. and Sahoo, L. (2022) 'Enhanced osmotic adjustment, antioxidant defense, and photosynthesis efficiency under drought and heat stress of transgenic cowpea overexpressing an engineered DREB transcription factor', Plant Physiology and Biochemistry, 193, pp. 1–13. Available at: https://www.elsevier.com/locate/plaphy Using inducible promotor DREB TF using stress inducible promotor for DREB transcription to overcome negative effect of it always being expressed – so drought response is only triggered in drought stress: Ramanathan, V., Muthurajan, R., Pralhad, S.M., Kambale, R., Madasamy, P., Shankarrao, C.N., Tamilselvan, S., Shillak, A.B., Nallathambi, J. and Rahman, H. (2021) ‘Controlled over-expression of AtDREB1A enhances tolerance against drought and salinity in rice’, Agronomy, 11(1), p. 159. Available at: https://doi.org/10.3390/agronomy11010159

Answer 10

Nelson et al PNAS (2007) http://www.ncbi.nlm.nih.gov/pubmed/17923671 to find this overexpressed 1500 factors in Arabidopsis Nelson, D.E., Repetti, P.P., Adams, T.R., Creelman, R.A., Wu, J., Warner, D.C., Anstrom, D.C., Bensen, R.J., Castiglioni, P.P., Donnarummo, M.G., Hinchey, B.S., Kumimoto, R.W., Maszle, D.R., Canales, R.D., Krolikowski, K.A., Dotson, S.B., Gutterson, N., Ratcliffe, O.J. and Heard, J.E. (2007) ‘Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-limited acres’, Proceedings of the National Academy of Sciences (PNAS), 104(42), pp. 16450–16455. Available at: https://doi.org/10.1073/pnas.0707193104

Answer 11

1. Overexpressed 1500 T FS in Arabidopsis 2.35S::AtNF-Y B plants showed drought tolerance 3. Micro-array. Limited targets (CCAAT). Polysaccharide metabolism. No obvious stress genes. 4. Maize orthologue identified. Overexpressed in maize, improved performance. NF-Y B forms a heterotrimer with he A and C subunits of transcriptional activator. This type of TF exists across most eukaryotes and typically act in concert with other regulatory factors to modulate gene expression. In this case, targets of NF-Y B overexpression tended to be genes with the promoter motif CCAAT. The microarray was performed in order to see if the genes affected in the overexpressers were the same as genes known to be associated with drought tolerance. They didn’t seem to be. They also compared gene upregulated by NF-Y B with genes overexpressed in response to a drought-related transcription factor (CBF4- not be confused with CBF1,2,3) or ABA. The genes were mostly different ones to the usual drought or ABA-associated genes.

Answer 12

ATNF – YB1 mutant characteristics: *Improved chlorophyll content *stomatal conductance *leaf temperature *reduced wilting *maintenance of photosynthesis Transgenic maize plants in greenhouse and field have visually observable improved drought tolerance. In both photographs, controls are in the left flat or row, and transgenics expressing ZmNF-YB2 are in the right flat or row. Three transgenic maize lines demonstrate improved yield in 2 years of yield testing Grey or black bars). Values plotted are increase on a percentage basis of transgenics over controls. All differences plotted are significant at P < 0.1. Data from three independent lines are shown with side-by-side comparison of 2 years' results. Base yield (yield of controls) was 4.6 metric tons/hectare (74 bushels/acre) in Year 1 and 6.4 metric tons/hectare (102 bushels/acre) in Year 2. Later explored in Canola (oil seed rape) see Xu et al Plos One 2014 http://www.ncbi.nlm.nih.gov/pubmed/25356551 And in Soybean Sun et al.