Periodic Reporting for period 4 - LUSH SPIKE (Genetic and Molecular Determinants of Spikelet Survival in Cereal Crops)
Reporting period: 2021-01-01 to 2021-06-30
Meeting the forecasted world demand for food remains a crucial challenge for plant scientists in this century. One promising avenue for improving grain yield of cereal crops, including wheat and barley, involves reducing spikelet mortality. Spikelets, the grain-bearing units of cereal spikes, usually form in excess and subsequently abort during development; increased spikelet survival is linked to increased numbers of grains per spike. Therefore, reducing spikelet mortality is an intriguing approach to improve grain yield.
In barley, the number of spikelets per spike at the awn primordium (AP) stage represents the maximum yield potential per spike. After the AP stage, significant spikelet mortality results in fewer grains per spike. Our previous results clearly indicated that spikelet survival in barley is highly genetically controlled (broad-sense heritability >0.80) and that the period from AP to tipping represents the most critical pre-anthesis phase related to spikelet reduction and grain yield per spike. However, the underlying genetic and molecular determinants of spikelet survival remain to be discovered. I therefore conducted an ambitious research program with an emphasis on using available genetic resources.
Our specific aims during the LUSH SPIKE project were to:
(i) discover quantitative trait loci (QTL) for spikelet survival and grain number per spike and validate these QTL in bi-parental doubled-haploid mapping populations,
(ii) isolate and functionally characterize Mendelized QTL using a map-based approach,
(iii) reveal gene regulatory networks determining spikelet survival during the critical spike growth period from AP to heading, and
(iv) elucidate spatio-temporal patterns of metabolite and phytohormone distributions in spike and spikelet sections during the critical growth period, using mass spectrometric imaging.
The results from LUSH SPIKE will advance our understanding of how to improve yields of cereal crops.
In barley, the number of spikelets per spike at the awn primordium (AP) stage represents the maximum yield potential per spike. After the AP stage, significant spikelet mortality results in fewer grains per spike. Our previous results clearly indicated that spikelet survival in barley is highly genetically controlled (broad-sense heritability >0.80) and that the period from AP to tipping represents the most critical pre-anthesis phase related to spikelet reduction and grain yield per spike. However, the underlying genetic and molecular determinants of spikelet survival remain to be discovered. I therefore conducted an ambitious research program with an emphasis on using available genetic resources.
Our specific aims during the LUSH SPIKE project were to:
(i) discover quantitative trait loci (QTL) for spikelet survival and grain number per spike and validate these QTL in bi-parental doubled-haploid mapping populations,
(ii) isolate and functionally characterize Mendelized QTL using a map-based approach,
(iii) reveal gene regulatory networks determining spikelet survival during the critical spike growth period from AP to heading, and
(iv) elucidate spatio-temporal patterns of metabolite and phytohormone distributions in spike and spikelet sections during the critical growth period, using mass spectrometric imaging.
The results from LUSH SPIKE will advance our understanding of how to improve yields of cereal crops.
Ad i+ii.) We phenotypically analyzed in total 29 genotypes of spring barley (all of which are parents of our DH mapping populations) under controlled growth (i.e. greenhouse, GH) and field conditions for their growth curve patterns. We show that the spikelet abortion process can be divided into several phases regardless of growth conditions (Thirulogachandar et al. 2021). For QTL discovery, we selected a diverse GWAS panel of six-rowed barleys (~400 accessions) based on high genotypic diversity; all accessions were selected for photoperiod sensitivity alleles at the major photoperiod locus Ppd-H1. In 2018, we already had 275 out of the ~400 accessions in a field trial (double rows per accession) and investigated traits such as maximum yield potential, spikelet survival, grain number per spike and other agronomically relevant characters for initial GWAS analyses (Kamal et al. 2022). QTL were validated in field seasons 2018 to 2020 using the whole panel of ~400 accessions and more markers (i.e. ~22 Mio from 2-3x genome coverage; following a whole-genome shotgun, WGS, approach). Moreover, 358 accessions have undergone detailed phenotypic analyses under controlled growth conditions (GH) during the fall/winter/early spring periods of the years 2018-19 and 2019-20. For both environments, field and GH, we found highly significant QTLs almost to single-gene resolution. Functional gene validation work is underway for three QTL regions (Huang et al.; in preparation/ Kamal et al.; in preparation).
Ad iii.) From our previous works related to the generation of a barley spike transcript atlas using laser-aided micro dissections of meristems (Thiel, Koppolu et al. 2021), we discovered that we had already covered the critical period for the initiation of spikelet abortion. Visible spikelet abortion is rather the end point of an occurring mitotic arrest of the apical inflorescence meristem, which starts on the cellular level much earlier. We therefore found transcript signatures for the onset of abortion/mitotic arrest in our available data set. Established GRNs are still under continuous investigations (Huang et al.; in preparation).
Ad iv.) To improve our understanding of hormonal and metabolite regulation of yield-establishing processes, such as spikelet decline, we established within the LUSH SPIKE project a detailed histological and microscopic analysis using the latest mass spectrometric imaging techniques (Peukert et al. 2014; Plant Cell) to integrate metabolite and hormonal analyses with organ growth over the critical period of development. Using such MALDI-MS imaging, we found for the first time the spatio-temporal distribution of important metabolites, such as sugars, amino acids and the phytohormone melatonin in developing barley spikes. By combining metabolomic, transcriptomic, and genetic approaches we show that apical abortion is associated with sugar depletion, amino acid degradation, and ABA biosynthesis and signaling. Senescence and defense-responsive transcription factor families, viz., NACs, HD-ZIPs, bZIPs, and MYBs, are amongst the putative candidate genes responsible for apical abortion. CRISPR/Cas9-mediated knock-out of barley GRASSY TILLERS1 (HvGT1) encoding an HD-ZIP transcription factor delayed apical abortion, thereby increasing the final spikelet number. We thus propose that modifying apical spikelet abortion by exploiting the identified putative regulators may help increase yield potential in barley and other related cereals (Shanmugaraj et al.; in preparation).
Ad iii.) From our previous works related to the generation of a barley spike transcript atlas using laser-aided micro dissections of meristems (Thiel, Koppolu et al. 2021), we discovered that we had already covered the critical period for the initiation of spikelet abortion. Visible spikelet abortion is rather the end point of an occurring mitotic arrest of the apical inflorescence meristem, which starts on the cellular level much earlier. We therefore found transcript signatures for the onset of abortion/mitotic arrest in our available data set. Established GRNs are still under continuous investigations (Huang et al.; in preparation).
Ad iv.) To improve our understanding of hormonal and metabolite regulation of yield-establishing processes, such as spikelet decline, we established within the LUSH SPIKE project a detailed histological and microscopic analysis using the latest mass spectrometric imaging techniques (Peukert et al. 2014; Plant Cell) to integrate metabolite and hormonal analyses with organ growth over the critical period of development. Using such MALDI-MS imaging, we found for the first time the spatio-temporal distribution of important metabolites, such as sugars, amino acids and the phytohormone melatonin in developing barley spikes. By combining metabolomic, transcriptomic, and genetic approaches we show that apical abortion is associated with sugar depletion, amino acid degradation, and ABA biosynthesis and signaling. Senescence and defense-responsive transcription factor families, viz., NACs, HD-ZIPs, bZIPs, and MYBs, are amongst the putative candidate genes responsible for apical abortion. CRISPR/Cas9-mediated knock-out of barley GRASSY TILLERS1 (HvGT1) encoding an HD-ZIP transcription factor delayed apical abortion, thereby increasing the final spikelet number. We thus propose that modifying apical spikelet abortion by exploiting the identified putative regulators may help increase yield potential in barley and other related cereals (Shanmugaraj et al.; in preparation).
1) We discovered that spikelet abortion in cereals is a predetermined developmental program (of the main culm) that is unexpectedly linked with the circadian clock, plastid differentiation and cell division of the inflorescence; all processes seem interlinked and show some level of plasticity depending on growth conditions (papers in revision and in preparation).
2) We will provide the first MALDI-MS images of developing inflorescences of cereals (i.e. barley), in which we show over time, e.g. chlorophyll biogenesis patterning, amino acid or sugar distributions. MALDI-MS in such a context can be considered as a breakthrough technology by providing a lot of spatial information (~10 μm), in particular where metabolites are located during cereal inflorescence growth and development. (paper in revision for the method development; paper in preparation for the biology)
3) We revealed that spikelet abortion can be initiated at variable developmental time points during inflorescence growth and development; it is indicative following the ‘spikelet stop’ approach. (paper: Venkatasubbu, Schnurbusch (2021) ‘Spikelet stop’ determines the maximum yield potential stage in barley. J Exp Bot, https://doi.org/10.1093/jxb/erab342)
4) We showed that different spike row-types of barley follow different strategies for grain number determination. Increasing grain numbers might be possible by augmenting potential spikelet numbers in two-rowed genotypes, while for six-rowed genotypes, spikelet survival needs to be improved. (Venkatasubbu et al. (2021) Strategies of grain number determination differentiate barley row-types. J Exp Bot, https://doi.org/10.1093/jxb/erab395)
2) We will provide the first MALDI-MS images of developing inflorescences of cereals (i.e. barley), in which we show over time, e.g. chlorophyll biogenesis patterning, amino acid or sugar distributions. MALDI-MS in such a context can be considered as a breakthrough technology by providing a lot of spatial information (~10 μm), in particular where metabolites are located during cereal inflorescence growth and development. (paper in revision for the method development; paper in preparation for the biology)
3) We revealed that spikelet abortion can be initiated at variable developmental time points during inflorescence growth and development; it is indicative following the ‘spikelet stop’ approach. (paper: Venkatasubbu, Schnurbusch (2021) ‘Spikelet stop’ determines the maximum yield potential stage in barley. J Exp Bot, https://doi.org/10.1093/jxb/erab342)
4) We showed that different spike row-types of barley follow different strategies for grain number determination. Increasing grain numbers might be possible by augmenting potential spikelet numbers in two-rowed genotypes, while for six-rowed genotypes, spikelet survival needs to be improved. (Venkatasubbu et al. (2021) Strategies of grain number determination differentiate barley row-types. J Exp Bot, https://doi.org/10.1093/jxb/erab395)