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Project titleStudy of the key players required for connecting ABA signaling and ABA-mediated drought and thermotolerance

AffiliationFederal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch of the Russian Academy of Sciences,

Research area 04 - BIOLOGY AND LIFE SCIENCES, 04-209 - Biotechnology (including biological nanotechnology)

KeywordsABA, ABA signaling, ABA-HSP/chaperone protein network, Drought Tolerance, Heat Stress Factor, Heat Stress Protein, Thermotolerance

Annotation
Heat stress response (HSR) is a conserved mechanism developed to increase the expression of heat shock proteins (HSPs) via a heat shock factor (HSF)-dependent mechanism. Signaling by the stress phytohormone abscisic acid (ABA) is involved in acquired thermotolerance as well. Relations of the ABA-mediated signaling pathway with other plant defense systems are poorly understood. This proposal is aimed at understanding how plant cells can integrate different pathways to tolerate high temperatures, drought and other stresses. The analysis will be performed within the ABA-HSP/chaperone protein network, mainly with the SnRK2s-ABF/AREBs-DREBs and SnRK2s-ABF/AREBs-HSFA6a signaling cascades as well as with recently identified members of zinc-finger proteins (ZF3) and RING E3 ligases (HIRPs), playing a vital role in the ABA-mediated thermotolerance. Proteomics and bioinformatics experiments would also allow obtaining new data about the ABA-HSP/chaperone signaling. These data will be confirmed by non-targeted and targeted proteomic experiments followed by bioinformatics investigation. The data on protein networks combining components of ABA signaling and HSF/ZF3/RING E3 ligase signaling will be obtained. Thus, this study would broaden our knowledge in understanding the complexity of this new ABA-signaling pathway integrated into the complex HS response network, and bring possible clues for improvement of temperature tolerance of plants. Knowledge of key points of cooperative influence of different defense pathways would allow crop protection and improvements by understanding the principles of growth arrest in stress-resistant plants. Modification of these signaling cascades will be carried out by the method of genomic editing. This application does not include genomic editing of crops; all works will be performed on Arabidopsis. We are building the future technological line CRISPR-cas9 in the form of Arabidopsis → crop cultivation consciously, realizing that at this stage, a model plant only gives a chance to understand the conjugacy of the networks. The work will be performed with SnRK2, namely the OST1/SnRK2.6-HOS1 module; genomic editing of this module will be carried out for the first time. Recent studies show the promise of modifying this module for crops, and this is now an important trend in agricultural biotechnology. Specific objectives of this project: 1. Understanding the reasons for switching to hermotolerance in modules SnRK2s-ABF/AREBs-DREBs and SnRK2s-ABF/AREBs-HSFA6a. 2. Evaluation of the role of zinc-finger proteins and RING E3 ligases in the regulation of these modules. 3. Creation of a CRISPR-cas9 modified Arabidopsis plants with increased stress resistance. 4. Finding nodes in the ABA-HSP/chaperone network, the impact on which will increase thermotolerance, but will not lead to growth retardation and developmental anomalies.

Expected results
The goal of this study is to realize a combined approach for investigation of the most important components of plant resistance to heat and other stresses. The combined approach includes molecular biology and biochemical methods of investigation, as well as genetic engineering and system biology methods. The synergistic effect necessary for the development of new bioengineering strategies is achieved. Although the study is performed with a model plant, its results will be further applied to crops. In this work, particular problems will be solved: - understanding the reasons for switching to thermotolerance in modules SnRK2s-ABF/AREBs-DREBs and SnRK2s-ABF/AREBs-HSFA6b. -evaluation of the role of zinc-finger proteins and RING E3 ligases in the regulation of these modules. - creation of a CRISPR-cas9 modified Arabidopsis plants with increased stress resistance. - finding nodes in the ABA-HSP/chaperone network, the impact on which will increase thermotolerance, but will not lead to growth retardation and developmental anomalies. A number of gene-edited Arabidopsis plants (Col-0) plants with significantly changed ABA responsiveness will be obtained. These plants will be tested for resistance to cold, high temperature, drought and salt. Enhancement of OST1/SnRK2.6 activity could modulate cold tolerance via the well-known ICE-CBF/DREB1 cascade or affects OST1-ABF2/AREB1-DREB1A pathway, whereby improving cold acclimation and drought resistance. The activation of OST1 may potentially affect thermotolerance via OST1-ABF2/AREB1-DREB2A or OST1-ABF2/AREB1-HSFA6b interactions. The most interesting plants showing prominent resistance to any kind of abiotic stress will be subjected to proteomic analysis. We expect this should results in identification of ABA signaling components involved, such as PYR/PYL/RCAR, ABI-clade protein phosphatases(PP)2Cs, SnRK2 protein kinases and ABF/AREB/ABI5 clade, CIPKs/CBLs, zinc-finger proteins ZF and RING E3 ligases, ABA-associated chromatin remodeling factors, and others; heat-shock proteins and associated factors HSP70s of DnaK and Hsp110/SSE subfamilies, HSP90s, HS factors, ABA-responsive CHIP and chaperonins. The planned results correspond to the world level of research. An attempt to connect two signaling systems to one network will be carried out for the first time. CRISP-Cas9 genomic editing of OST1 / SnRK2.6-HOS1 will be held for the first time.

Annotation of the results obtained in 2020
A network of protein-protein interactions of the signaling systems of abscisic acid (ABA) and brassinosteroids (BR) was built, which made it possible to explain the “phenomenon of the brassinosteroid receptor BRL3”. Fabregas et al. (2018) in Nature Communication reported that overexpression of the vascular brassinosteroid receptor BRL3 provides improved drought tolerance without disrupting plant growth. We summarized recent evidence of BR and abscisic acid (ABA) signaling and found that the BRL3-mediated drought tolerance phenomenon can be attributed to stress memory generation (a process known as “priming” or “acclimatization”). Stress memory can be realized by signaling through the cascade BRL3 → BSK1 / 3 → BSU1 2 BIN2 → BSK1 / 3, where BIN2 integrates the BR and ABA pathways. As expected, HOS1 knockout provided an increase in cold resistance in all hos1cas9 Arabidopsis lines tested, without inhibiting growth. This effect appears to be due to a decrease in ICE1 inhibition in the SnRK2.6/OST1-HOS1-ICE1 cold resistance module, which is in line with the theoretical predictions. Gene expression analysis showed that the net effect of suppressing HOS1 was the activation of DREB1A and DREB2C. These proteins provide increased resistance to cold and heat stress, respectively. DREB gene activation may partially explain the cold and thermal resistance of mutant lines. In the ABA signal transduction chain, the genes of almost all components were activated, such as the ABA PYL4 receptor, PP2Cs HAI1, ABI1 and ABI2 phosphatases, OST1 / SnRK2.6, SnRK2.2 and SnRK2.3 kinases, transcription factors ABI4, ABI5, DREB1A, DREB2A and DREB2C. Apparently, inhibition of HOS1 activity created conditions for more active functioning of the ICE-CBF / DREB pathway. This pattern resembles moderate activation of the ABA pathway under stress. The mutant lines also acquired increased resistance to elevated temperatures. An important circumstance that could lead to an increase in the temperature resistance of hos1cas9 mutant lines is a significant activation of genes encoding heat shock factors (HSF), namely HsfA6a and HsfA2. In hos1cas9 mutant lines, the biosynthesis of secondary metabolites is activated. The most significant increase was observed for flavonoids (derivatives of quercetin, kaempferol and isoametin, 1.2-4.2 times) in plants and 1-methoxyindol-3-ylmethyl GS (20-60 times) and 4-hydroxyindol-3-ylmethyl GS (2.8 to 3.6 times) in callus cultures. The increase, apparently, was associated with the stimulation of de novo biosynthesis, as shown by the analysis of the expression of genes encoding transcription factors and biosynthetic enzymes. Expression patterns of key regulatory genes of the ABA pathway, heat shock factors, and other pathways in hos1cas9 lines indicate a partial activation of ABA-dependent and ABA-independent components, including the activation of the chaperone pathway and some elements of immunity, including those affecting secondary metabolism.

Publications

1. - CRISPR/Cas9-Mediated Knockout of HOS1 Reveals Its Role in the Regulation of Secondary Metabolism in Arabidopsis thaliana Plants (Basel), 2021 Jan 6;10(1):104 (year - ) https://doi.org/10.3390/plants10010104

2. G.N. Veremeichik, Y.N. Shkryl, T.Y. Gorpenchenko, S.A. Silantieva, T.V. Avramenko, E.V. Brodovskaya, V.P. Bulgakov Inactivation of the auto-inhibitory domain in Arabidopsis AtCPK1 leads to increased salt, cold and heat tolerance in the AtCPK1-transformed Rubia cordifolia L cell cultures Plant Physiology and Biochemistry, - (year - 2020)

3. Victor P Bulgakov, Tatiana V Avramenko Linking Brassinosteroid and ABA Signaling in the Context of Stress Acclimation INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 2020 Jul 20;21(14):5108 (year - 2020) https://doi.org/10.3390/ijms21145108

4. Wu HC, Bulgakov VP, Jinn TL. Pectin Methylesterases: Cell Wall Remodeling Proteins Are Required for Plant Response to Heat Stress. Front Plant Sci., 9:1612 (year - 2019)

Annotation of the results obtained in 2018
1. The first review article presents a connection of signals of abscisic acid (ABA) and heat shock factors (HSF) with cell wall remodeling during cold and heat stress. The association of HSF with pectin methyltransferases and methylesterases, as well as with induced by heat stress [Ca2+] cyt = nuc oscillations, as well as calmodulins, is shown. The role of all these factors in the regulation of stomatal apertures under various types of stress is shown, as well as calmodulin. The role of all these factors in the regulation of stomatal apertures under various types of stress is also shown. Hui-Chen Wu, Victor P. Bulgakov, Tsung-Luo Jinn. Pectin Methylesterases: Cell Wall Remodeling Proteins Are Required for Plant Response to Heat Stress. Frontiers in Plant Science, 2018, V.9, Article 1612, https://doi.org/10.3389/fpls.2018.01612 IF 4,299, Q1. https://www.frontiersin.org/articles/10.3389/fpls.2018.01612/full?utm_source=FRN&utm_medium=EMAIL_IRIS&utm_campaign=EMI_XIA_181116_Milestones_Followers_article_page 2. In the second review article, the idea of the need to find the elements linking the regulatory mechanisms of ABA and chaperones is implemented. The abscisic acid (ABA) and chaperone signaling pathways are the central regulators of plant stress defense. Despite their significance and potential overlap, these systems have been described separately. We summarize information about mechanisms by which the ABA and chaperone signaling pathways might be co-regulated, taking into account the idea that these defense systems must work in a coordinated manner. The central factors that join the ABA and chaperone signaling systems are the SWI/SNF chromatin remodeling proteins, such as BRM, SWI3B and SWI3C, all of which are involved in stress memory. The signaling module at the crosstalk between ABA and heat-shock factor (HSF) signaling has a core structure: SnRK2s/PP2Cs/ABRE–binding factors–BRM/SWI3B/SWI3C–HSFs. Peptidyl-prolyl cis-trans isomerases seem to play an accessory role in pathway coordination. A benefit from coordination is that the signals sensed through both the ABA and chaperone signaling systems are perceived and stored via chromatin-remodeling factors. We propose new bioengineering strategies, which we term ‘bioengineering memory’, for improving plant stress resistance. Victor P. Bulgakov, Hui-Chen Wu, and Tsung-Luo Jinn. Coordination of abscisic acid (ABA) and chaperone signaling in plant abiotic stress responses: emerging perspectives of ‘bioengineered memory’. Biotechnology Advances (under review), IF 11.452, Q1. 3. Using online services (E-CRISP, CRISPR Design), optimal RNA guides were selected to the first and second Arabidopsis exon HOS1 (HOS1-1 and HOS1-2, respectively), taking into account the existence of two splice gene variants (NM_129540.5 и NM_001336798.1). RNA guides to the first exons of the HsfA7a and HsfA7b genes have been developed. 4. The binary vectors pPZP-RCS2-nptII-Cas9-HOS1-1, pPZP-RCS2-nptII-Cas9-HOS1-2, pPZP-RCS2-nptII-Cas9-HSFA7A and pPZP-RCS2-nptII-Cas9-HSFA7B were obtained. These vectors are transferred to the hyper-virulent Agrobacterium tumefaciens EHA105 strain for plant transformation. 5. Six plants transformants Cas9-HOS1-2 were obtained. The true transformation efficiency (RTE) was 2%. It was established also two plants Cas9-HOS1-2, which not contain T-DNA. 6. Genetic constructs for HDR-mediated activation of the OST1 gene were obtained. The pPZP-RCS2-nptII-Cas9-OST1-HOS1-1 and pPZP-RCS2-nptII-Cas9-OST1-HOS1-2 constructs were transferred to A. tumefaciens EHA105 for plant transformation. T1 plants obtained. 7. T1 plants Cas9-HSFA7A and Cas9-HSFA7B were obtained. 8. In constructs for genomic editing, the regulatory elements of the nptII gene were replaced with the promoter and terminator of the 35S CaMV gene in order to increase the efficiency of the selective marker.

Publications

1. Bulgakov VP The rolB plant oncogene affects multiple signaling protein modules related to hormone signaling and plant defense. Scientific Reports, 8(1):2285 (year - 2018) https://doi.org/10.1038/s41598-018-20694-6

Annotation of the results obtained in 2019
Two major stress defense systems of plants, such as the abscisic acid (ABA) signaling pathway and chaperone signaling are the central regulators of stress defense. Despite their significance, these systems have been described separately. In this review, we summarized information about mechanisms, by which the ABA and chaperone signaling pathways might be co-regulated, taking into account the idea that these defense systems must work in a coordinated manner. The central factors joining ABA and chaperone signaling systems are the SWI/SNF chromatin remodeling proteins, such as BRM, SWI3B and SWI3C, involved in stress memory. The signaling module at the crosstalk between ABA and heat-shock factor (HSF) signaling has a core structure: SnRK2s/PP2Cs/ABRE-binding factors-BRM/SWI3B/SWI3C-HSFs. Peptidyl-prolyl cis-trans isomerases seem to play an accessory role in the pathways coordination. Benefit from the coordination is that the signals sensing both through ABA- and chaperone signaling systems perceived and stored via chromatin-remodeling factors. New bioengineering strategies, which we termed ‘bioengineering memory’, were proposed for improvement of plant stress resistance. Bulgakov V.P., Wu H.C., Jinn T.L Coordination of ABA and Chaperone Signaling in Plant Stress Responses. Trends in Plant Science 2019 Jul;24(7):636-651. IF 14.004 Q1 https://www.sciencedirect.com/science/article/pii/S1360138519301001?via%3Dihub

Publications

1. Bulgakov V.P., Wu H.C., Jinn T.L. Coordination of ABA and Chaperone Signaling in Plant Stress Responses Trends in Plant Science, 24(7):636-651 (year - 2019) https://doi.org/10.1016/j.tplants.2019.04.004

2. Shkryl Y.N., Veremeichik G.N., Silantieva S.A., Bulgakov V.P. Differential expression of calcium-dependent protein kinase genes Plant Gene, - (year - 2019) https://doi.org/10.1016/j.plgene.2019.100215

3. - Стало известно, как растения запоминают пережитые экстремальные условия, чтобы приспособиться к ним РНФ, 22 мая 2019 г. (year - )

4. - Стало известно, как растения запоминают пережитые экстремальные условия, чтобы приспособиться к ним Газета.ru, 21.05.2019 (year - )

5. - Ученые: растения защищаются от экстремальных условий с помощью "памяти к стрессам" SakhaNews, 22.05.2019 (year - )

6. - Учёные ДВФУ и ДВО РАН: как снизить потери урожая в жаре, холоде и засухе ДВФУ Новости, 29 мая 2019 - Наука и инновации (year - )

7. - Bioengineers suggested ways to reduce crop losses caused by heat, cold and drought EurecAlert American Association for the Advancement of Science, NEWS RELEASE 28-MAY-2019 (year - )

8. - Растения запоминают пережитые экстремальные условия, чтобы приспособиться PLANET TODAY, 22 Мая 2019 (year - )

9. - Как растения запоминают стрессы Наука и Жизнь, 25 мая 2019, № 6 (year - )

10. - Ученые: растения защищаются от экстремальных условий с помощью "памяти к стрессам" SELDON NEWS, 22 мая 2019 г (year - )

11. - Scientists demonstrate plant stress memory and adaptation capabilities PHYS ORG, MAY 31, 2019 (year - )

12. - Стрессоустойчивость растений планируют повысить с помощью «биоинженерии памяти» Indicator, 22 мая 2019 (year - )

13. - Стрессоустойчивость растений планируют повысить с помощью «биоинженерии памяти» Российская Академия Наук -Новости, 22.05.2019 (year - )

14. - Стрессоустойчивость растений планируют повысить с помощью «биоинженерии пямяти» Об этом сообщает "Рамблер". Далее: https://news.rambler.ru/scitech/42211876/?utm_content=news_media&utm_medium=read_more&utm_source=copylink РАМБЛЕР, 21 мая 2019 (year - )