SEEDs for the FUTURE: developing barley lines with improved yield and grain quality under drought stress during seed filling.
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GABI-GRAIN is a BMBF-funded research project (2007 – 2010) that is carried out in tight cooperation between several academic institutes and industrial partners:
- Martin Luther University of Halle-Wittenberg (MLU)/Institute of Agriculture and Nutritional Sciences,
Drought is one of the major environmental constraints that severely limit crop productivity. Yield safety could be improved if future breeding attempts which include the new knowledge acquired on the processes determining developmental programs that enhance yield stability and improve grain quality under terminal drought.
The aim of this application-oriented project is to use barley as a model crop to explore natural genetic variation among (i) elite breeding material, (ii) barley core collection gene bank material and (iii) nearly 120 introgression lines covering two populations with wild barley accessions as donors in an attempt to understand mechanisms of reaching enhanced yield stability and uncompromised seed quality under drought stress during seed filling.
Expected deliverables are:(a)increased knowledge on regulatory networks underlying yield/quality parameters under environmental stress conditions to aid knowledge-based breeding with general relevance for the improvement of seed traits; (b) verified candidate genes for the development of different drought tolerant GMO crops; (c) trait-associated DNA and protein markers for marker-assisted selection (MAS) and (d) development of large F1 populations of DH barley lines as well as GM plants with modified ABA responses for minimized yield loss under drought stress. To achieve the above mentioned project aims five major modules were derived.
Module 1: Exploring natural genetic variation for improved yield and better remobilization capacities under drought stress during seed filling (IPK partners and NORDSAAT-SAATZUCHT) and production of large DH populations as a new resource of breeding material for improved seed quality under terminal drought (KWS LOCHOW)
Module 2: Identification of key metabolites, proteins and enzymes in the regulatory networks determining seed filling under drought (IPK partners, MLU and MPI-Golm)
Module 3: Identifying key regulators influencing broad adaptation and yield stability under drought stress through transcriptome analysis (IPK partners)
Module 4: Analysis of expression (e) QTLs and genetic diversity in candidate genes for grain yield and quality under drought (IPK partners)
Module 5: A transgenic strategy for improved seed filling under drought stress by altering ABA metabolism in seeds (IPK partners)
The following objectives are achieved from the above described 5 modules and the corresponding work packages.
(i) We explored the untapped genetic reserves of wild barley in BC3 double haploid introgression lines (DH ILs) , preselected gene bank and elite lines for improved seed yield and quality through field screening [WP1.1: L. Kuntze, NORDSAAT and A. Börner, IPK] and precise green house screening by implementing efficient physiological techniques [WP1.2 and 2.1 : N. Sreenivasulu and H. Rolletschek, IPK], to identify stable performing and susceptible lines through correlation and QTL approaches [WP1.2 and 4.4: N. Sreenivasulu and M. Röder, IPK]; including the second IL population [WP2.3: K. Pillen, MLU].
(ii) Using the 12 selected lines we identified key metabolites [WP2.1 and 2.2 : H. Rolletschek and N. Sreenivasulu, IPK] and enzymes [2.2 : M. Strickert and R. Sulpice, MPIMP] influencing optimized seed filling under drought and thereby identified the benefits of remobilizing and stay green mechanisms for optimum seed filling and unaltered seed quality.
(iii) We further revealed key candidate genes and regulatory networks underlying yield/quality parameters under drought [WP 3.1 and 3.2: N. Sreenivasulu, M. Strickert and U. Wobus, IPK]. Global relationships between transcripts, metabolites and enzymes were predicted to define the nodes of connections between upstream signalling networks with the downstream pathway genes influencing metabolic adjustment for acquainted drought tolerance during seed filling [WP 3.3: B. Usadel and M. Stitt, MPIMP and N. Sreenivasulu, IPK].
(iv) The second introgression line population (S42-ILs) that originate from the cross Scarlett (H. vulgare) xISR42-8 (H. spontaneum) containing 49 ILs were screened for drought tolerance during grain filling in the glasshouse, phenotyped and QTLs were derived. To understand the molecular mechanism controlling identified QTL a proteomics study is currently being performed from the 4 most interesting ILs.
(v) We developed 141 new SNP markers for key regulators and metabolic pathway genes influencing seed yield/quality under drought, haplotyped 20 selected candidate genes from starch and storage protein synhesis, identified 81 haplotypes [WP 4.1 and 4.2: M. Röder, IPK], developed two different populations [WP 4.3 in cooperation with V. Korzun KWS Lohow] and also validated key genes defined in WP3.2 by the estimation of eQTLs with a qRT-PCR platform [WP 4.4: M. Röder, IPK].
(vi) The derived knowledge from the complementary genomic approaches and the line selection results has been passed to KWS-Lochow to support the development of superior-performing lines by non-GMO approaches. [N. Sreenivasulu, IPK]. KWS Lochow developed F1 crosses and produced DH with a total of 4225 plants. The field trials were conducted during 2009 at two locations (Wohlde, Germany and Walewice, Poland, known to possess different precipitation rate), major agronomical characters (plant length, TGW, etc) and yield performances have been recorded and initial biostatistical analysis has been performed. The resulting F1 crosses and DH lines showed good differentiation for grain yield components from plant material grown in two locations with different precipitation rate [WP 1.3: V.Korzun, KWS-Lochow].
(vii) ABA-immunomodulated transgenic plants expressing anti-ABA-single chain Fv antibodies (using 3 different promoters such as ubiqutin, 1AX1 and drought-induced LEA19.3) were produced through Agrobacterium transformation, the T0 plants screened and DH lines produced by anther culture and the performance of DH lines assessed for seed quality under control conditions [WP 5.1: U. Conrad, J.Kumlehn and W. Weschke, IPK]. ABA-immunomodulated transgenic plants showed altered hexose/sucrose ratio with induction of storage proteins in endosperm fraction but did not show the benefits for improved yield under control and drought stress treatments due to substantial elevated ABA levels in the developing seed.
In addition, the key target genes from ABA biosynthesis, degradation and signalling have been targeted for altered ABA content and ABA sensitivity through overexpression and RNAi approaches with 16 different constructs. Regenerated homozygous DH plants with stably inserted transgenes include 3 DH lines from SalT-NCED, 2 DH lines from Lea19.3-RPK and 1 DH line from SalT-RPK construct. In all those cases where we got no DH plants (1Ax1-NCED and Lea19.3-NCED), homozygous lines were identified from the T1 generation by traditional segregation analysis. The DH & T2 transgenic plants together with WT (Golden promise) plants were subjected to drought treatment during seed filling in green house. The SalT-NCED DH transgenic lines and Lea19.3-NCED T2 transgenic plants confirm the only minor reduction of TGW under stress by being highly water use efficient with less reduction of assimilation and chlorophyll b content under drought. Very interestingly, among all the transgenic plants studied, the SalT-NCED DH lines showed spike branching resulting in double spikes per tiller (line SN 412: up to 60% and line SN410: 40%) with minor percentages of up to 3 to 5 spike branching. All these selected lines have been grown again under field like conditions by sowing them directly on soil within small plots (rain shelters) and confirmed the trends of positive benefits of improved yield stability under drought. The selected lines have been characterized at physiological, biochemical and transcriptome level to reveal the mechanisms. The SalT-NCED DH transgenic lines and Lea19.3-NCED T2 transgenic plants showed elevation of ABA in flag leaves under short term drought stress and eventually the ABA levels have been brought back to nearly basal levels under prolonged drought treatments. On the contrary, wild type plants do not respond so quickly to elevate ABA and over the long term drought stress ABA levels were maintained at substantially higher levels. From these results we conclude that maintaining ABA homeostasis under terminal drought is an important target for the reprogramming of seed metabolism to promote optimum seed yield and quality under terminal drought [WP 5.2 and WP 5.3: N. Sreenivasulu, J.Kumlehn and U. Wobus, IPK].
Publications of GABI-GRAIN Project:
1. Govind, G., Seiler, C., Wobus, U. and Sreenivasulu, N. Importance of ABA homeostasis under terminal drought stress in regulating grain filling events. Plant Signaling and Behavior (in press).
2. Lohse, M., Nunes-Nesi, A., Osorio, S., Krüger, P., Childs, L., Hannemann, J., Nagel, A., Walther, D., Selbig, J., Sreenivasulu, N., Stitt, M., Fernie, A.R. and Usadel, B. Robin: An intuitive wizard application for R-based microarray quality assessment and analysis. Plant Physiology 153: 642-651, 2010.
3. Melkus, G., Rolletschek, H., Fuchs, J., Radchuk, V., Grafahrend-Belau, E., Sreenivasulu, N., Rutten, T., Weier, D., Heinzel, N., Schreiber, F., Altmann, T., Jakob, P.M. and Borisjuk, L. Dynamic 13C⁄1H NMR imaging uncovers sugar allocation in the living seed. Plant Biotechnology Journal (in press)
4. Seiler, C., Harshavardhan, V.T., Rajesh, K., Stricker, M., Rolletschek, H., Scholz, U., Wobus, U. and Sreenivasulu, N: ABA biosynthesis and degradation contributing to ABA homeostasis during barley seed development under control and terminal drought stress conditions. Journal of Experimental Botany 62: 2615-2632, 2011.
5. Sreenivasulu, N., Borisjuk, L., Junker, B., Mock, H.-P., Rolletschek,H., Seiffert, U., Weschke, W. and Wobus, U. (2010) Barley grain development – towards an integrated view. International Review of Cell and Molecular Biology 281: 49-89, 2010.
6. Sreenivasulu N, Sunkar R, Wobus U, Strickert M: Array platforms and bioinformatic tools for the analysis of the plant transcriptome in response to abiotic stress. In: Methods in Molecular Biology 639: 71-93, 2010.
7. Sreenivasulu, N., Röder, M., and Wobus, U. Trockenstress – eine Suche nach den Ursachen und nach neuen Wegen zur Züchtung trockentoleranter Getreide. In: GENOMXPRESS. 4: pp. 4-6, 2010.
8. Sreenivasulu, N., Graner, A. and Wobus, U. Barley Genomics: An Overview, International Journal of Plant Genomics Volume 2008, Article ID 486258, 13 pages, 2008.
9. Sreenivasulu, N., Usadel, B., Winter, A., Radchuk, V., Scholz, U., Stein, N., Weschke, W., Strickert, M., Close, T. J., Stitt, M., Graner, A. and Wobus, U. Barley grain maturation and germination: Metabolic pathway and regulatory network commonalities and differences highlighted by new MapMan/PageMan profiling tools. Plant Physiology 146: 1738-1758, 2008.
10. Worch, S., Kalladan, R., Harshavardhan, V.T., Pietsch, C., Korzun, V., Kuntze, L., Börner, A., Wobus, U., Röder, M.S. and Sreenivasulu, N: A combined linkage and expression analysis of barley genes regulated by terminal drought stress influencing seed quality. BMC Plant Biology 11: 1, 2011.
11. Rajesh, K., Harshavardhan, V.T., Seiler, C., Strickert, M., Rolletschek, H., Korzun, V., Usadel, B., Sulpice, R., Stitt, M., Wobus, U. and Sreenivasulu, N: Regulatory networks determining barley stay-green and senescence/remobilization phenotypes and their influence on seed yield and quality under terminal drought. (Manuscript in preparation).
12. Harshavardhan, V.T. et al., Transgenic strategy to improve grain weight under terminal drought through altered ABA homeostasis. (Manuscript in preparation).