Green Revolution DELLAs: From translational reinitiation to future sustainable agriculture

The Green Revolution (GR) of the 1960s saw spectacular increases in cereal crop yields, enabled by widespread adoption of high-yielding semi-dwarf varieties and increased fertilizer use. Semi-dwarfism is caused by mutant wheat Reduced height-1 (Rht-1) and rice semi-dwarf1 (sd1) alleles, respectively, which confer increased harvest index (grain to straw ratio) and reduced yield loss from “lodging” (flattening of crops by wind or rain). However, despite their obvious global food security benefits, the underlying mechanisms via which GR dwarfing alleles actually work remained unknown. This began to change with the molecular discovery of Rht1 (Peng et al., 1999Peng J. Richards D.E. Hartley N.M. Murphy G.P. Devos K.M. Flintham J.E. Beales J. Fish L.J. Worland A.J. Pelica F. et al.“Green revolution” genes encode mutant gibberellin response modulators.Nature. 1999; 400: 256-261Crossref PubMed Scopus (1396) Google Scholar). Previous studies of Arabidopsis thaliana had discovered DELLA proteins (DELLAs), key components of the mechanism enabling the phytohormone gibberellin (GA) to promote growth. DELLAs are nuclear-localized growth inhibitors, and GA promotes plant growth by overruling their growth-inhibitory activity (Peng et al., 1997Peng J. Carol P. Richards D.E. King K.E. Cowling R.J. Murphy G.P. Harberd N.P. The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses.Genes Dev. 1997; 11: 3194-3205Crossref PubMed Scopus (828) Google Scholar). GA overrules DELLAs by targeting them for destruction by the ubiquitin-proteasome system (Sasaki et al., 2003Sasaki A. Itoh H. Gomi K. Ueguchi-Tanaka M. Ishiyama K. Kobayashi M. Jeong D.H. An G. Kitano H. Ashikari M. et al.Accumulation of phosphorylated repressor for gibberellin signaling in an F-box mutant.Science. 2003; 299: 1896-1898Crossref PubMed Scopus (481) Google Scholar). Analysis of the properties of the Arabidopsis thaliana gai (GA-insensitive) mutant was an important initial catalyst of these advances. Compared with wild-type (WT) GAI, the gai allele encodes a mutant gai protein (Figure 1A) lacking a 17 amino acid segment portion of the eponymous DELLA domain. The gai protein is resistant to GA-promoted proteasome-dependent destruction, and hence accumulates to confer genetically dominant semi-dwarfism (Peng et al., 1997Peng J. Carol P. Richards D.E. King K.E. Cowling R.J. Murphy G.P. Harberd N.P. The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses.Genes Dev. 1997; 11: 3194-3205Crossref PubMed Scopus (828) Google Scholar). The wheat GR Rht-B1b and Rht-D1b alleles, like Arabidopsis thaliana gai, also confer dominant GA-insensitive dwarfism. The molecular discovery of Rht-1 revealed that Rht-B1b and Rht-D1b, again like gai, are mutant DELLA-encoding alleles (Peng et al., 1999Peng J. Richards D.E. Hartley N.M. Murphy G.P. Devos K.M. Flintham J.E. Beales J. Fish L.J. Worland A.J. Pelica F. et al.“Green revolution” genes encode mutant gibberellin response modulators.Nature. 1999; 400: 256-261Crossref PubMed Scopus (1396) Google Scholar; see predicted encoded proteins compared with WT Rht-B1a in Figure 1A). Later, SD1 was shown to encode a GA biosynthetic GA20-oxidase, and rice GR sd1 allele to confer reduced GA20-oxidase activity, reduced bioactive GA levels, and consequent dwarfing accumulation of the rice DELLA protein SLR1 (Sasaki et al., 2002Sasaki A. Ashikari M. Ueguchi-Tanaka M. Itoh H. Nishimura A. Swapan D. Ishiyama K. Saito T. Kobayashi M. Khush G.S. et al.Green revolution: a mutant gibberellin-synthesis gene in rice.Nature. 2002; 416: 701-702Crossref PubMed Scopus (794) Google Scholar; Spielmeyer et al., 2002Spielmeyer W. Ellis M.H. Chandle P.M. Semidwarf (sd-1), "green revolution" rice, contains a defective gibberellin 20-oxidase gene.Proc. Natl. Acad. Sci. U S A. 2002; 99: 9043-9048Crossref PubMed Scopus (573) Google Scholar; Li et al., 2018Li S. Tian Y. Wu K. Ye Y. Yu J. Zhang J. Liu Q. Hu M. Li H. Tong Y. et al.Modulating plant growth-metabolism coordination for sustainable agriculture.Nature. 2018; 560: 595-600Crossref PubMed Scopus (200) Google Scholar). These advances had begun to solve the mystery of how GR genes work: GR alleles confer semi-dwarfism and increased yield through enhanced DELLA accumulation. Nevertheless, a major puzzle remained. The Rht-B1b and Rht-D1b open reading frames both contain premature stop codons shortly following the translation initiation AUG codon (Peng et al., 1999Peng J. Richards D.E. Hartley N.M. Murphy G.P. Devos K.M. Flintham J.E. Beales J. Fish L.J. Worland A.J. Pelica F. et al.“Green revolution” genes encode mutant gibberellin response modulators.Nature. 1999; 400: 256-261Crossref PubMed Scopus (1396) Google Scholar) (Figure 1A). At first sight these mutations might be expected to prevent translation into a functional protein. However, Rht-B1b and Rht-D1b are considered to be gain-of-function alleles that encode functional repressors of GA signaling. To explain this apparent discrepancy, Peng et al., 1999Peng J. Richards D.E. Hartley N.M. Murphy G.P. Devos K.M. Flintham J.E. Beales J. Fish L.J. Worland A.J. Pelica F. et al.“Green revolution” genes encode mutant gibberellin response modulators.Nature. 1999; 400: 256-261Crossref PubMed Scopus (1396) Google Scholar proposed that translational reinitiation at an alternative methionine start codon closely following the mutant stop codons could generate N-terminally truncated Rht-B1b and Rht-D1b proteins lacking the DELLA domain. Such truncated proteins would likely confer dominant semi-dwarfism, like other mutant DELLA domain lacking DELLAs (e.g., Arabidopsis gai; maize d8/mpl1; Peng et al., 1999Peng J. Richards D.E. Hartley N.M. Murphy G.P. Devos K.M. Flintham J.E. Beales J. Fish L.J. Worland A.J. Pelica F. et al.“Green revolution” genes encode mutant gibberellin response modulators.Nature. 1999; 400: 256-261Crossref PubMed Scopus (1396) Google Scholar). Although supported by the observation that a mutant stop codon disrupting sequence encoding a C-terminal region (named GRAS domain) of Rht-B1b suppresses dwarf phenotype (Mo et al., 2018Mo Y. Pearce S. Dubcovsky J. Phenotypic and transcriptomic characterization of a wheat tall mutant carrying an induced mutation in the C-terminal PFYRE motif of RHT-B1b.BMC Plant Biol. 2018; 18: 253Crossref PubMed Scopus (10) Google Scholar), the translational reinitiation hypothesis remained untested at the molecular level for many years. An important new report now provides a molecular test of this hypothesis (Van De Velde et al., 2021Van De Velde K. Thomas S.G. Heyse F. Kaspar R. Van Der Straeten D. Rohde A. N-terminal truncated RHT-1 proteins generated by translational reinitiation cause semi-dwarfing of wheat Green Revolution alleles.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.01.002Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). First, expression of constructs designed to express tagged versions of the proposed N-terminally truncated Rht-B1b protein in transgenic wheat confers semi-dwarfism. Second, this expression generates a truncated Rht-B1b protein that is resistant to GA-promoted degradation, and presumably confers the observed semi-dwarf phenotype. Further experiments test an alternative hypothesis, that Rht-B1b and Rht-D1b semi-dwarfing phenotypes are conferred by a putative short peptide initiated at the original start codon and terminating at the premature mutant stop codons (Figure 1A). Transgenic expression of immunologically detectable levels of this peptide had no detectable effect on plant height, indicating that this alternative hypothesis is unlikely to be correct. Thus, although translational reinitiation is presumably relatively inefficient, the N-terminally truncated DELLA protein of the first hypothesis is likely responsible for conferring Rht-B1b and Rht-D1b phenotypes. This study also solves another puzzle. GA promotes seed germination by overcoming dormancy and stimulating aleurone secretion of the α-amylase enzyme that mobilizes endosperm starch reserves. In consequence, expression of gai in transgenic rice confers not only GA-insensitive dwarfism, but also blocks the GA-induced aleurone α-amylase response (Fu et al., 2001Fu X. Sudhakar D. Peng J J. Richards D.E. Christou P. Harberd N.P. Expression of Arabidopsis GAI in transgenic rice represses multiple gibberellin responses.Plant Cell. 2001; 13: 1791-1802Crossref PubMed Scopus (85) Google Scholar). Similarly, the severely dwarfing wheat Rht-B1c allele, which encodes a mutant protein containing a 30 amino acid insertion blocking the destruction of DELLA, and which, because of its severity, has not been used in wheat breeding, also confers increased dormancy and reduced GA-induced α-amylase activity (Van De Velde et al., 2021Van De Velde K. Thomas S.G. Heyse F. Kaspar R. Van Der Straeten D. Rohde A. N-terminal truncated RHT-1 proteins generated by translational reinitiation cause semi-dwarfing of wheat Green Revolution alleles.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.01.002Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). In contrast, the Rht-B1b and Rht-D1b alleles cause dwarfism without affecting either seed dormancy or α-amylase response (Van De Velde et al., 2021Van De Velde K. Thomas S.G. Heyse F. Kaspar R. Van Der Straeten D. Rohde A. N-terminal truncated RHT-1 proteins generated by translational reinitiation cause semi-dwarfing of wheat Green Revolution alleles.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.01.002Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). How could these discrepancies be resolved? This new report indicates that they are due to differential regulation of translational reinitiation in shoots and aleurone. Translational reinitiation generates N-terminally truncated DELLAs in Rht-B1b and Rht-D1b shoots, thus conferring dwarfism, while lack of translational reinitiation in aleurone cells permits the GA-induced α-amylase response. Conversely, Rht-B1c acts independently of translational reinitiation and confers a phenotype in both shoot and aleurone (Van De Velde et al., 2021Van De Velde K. Thomas S.G. Heyse F. Kaspar R. Van Der Straeten D. Rohde A. N-terminal truncated RHT-1 proteins generated by translational reinitiation cause semi-dwarfing of wheat Green Revolution alleles.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.01.002Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Semi-dwarf GR varieties require a relatively high level of inorganic nitrogen (N) fertilizer input to achieve high grain yield. Despite the undeniable food security benefits of the GR, the associated high N inputs have caused extensive environmental damage. A sustainable post-GR agriculture must therefore build upon the advances of the GR by reducing N fertilizer use, while also maintaining or increasing grain yield. GR varieties actually have reduced N-mediated growth responses and N-use efficiency (NUE), because the accumulation of mutant wheat Rht-B1b or Rht-D1b or rice SLR1 impacts on balancing multi-level interactions with the GRF4 and NGR5 proteins (Li et al., 2018Li S. Tian Y. Wu K. Ye Y. Yu J. Zhang J. Liu Q. Hu M. Li H. Tong Y. et al.Modulating plant growth-metabolism coordination for sustainable agriculture.Nature. 2018; 560: 595-600Crossref PubMed Scopus (200) Google Scholar; Wu et al., 2020Wu K. Wang S. Song W. Zhang J. Wang Y. Liu Q. Yu J. Ye Y. Li S. Chen J J. et al.Enhanced sustainable green revolution yield via nitrogen-responsive chromatin modulation in rice.Science. 2020; 367: eaaz2046Crossref PubMed Scopus (85) Google Scholar). The new knowledge that Rht-B1b and Rht-D1b encode N-terminally truncated DELLAs that are active in shoot but not in grain (Van De Velde et al., 2021Van De Velde K. Thomas S.G. Heyse F. Kaspar R. Van Der Straeten D. Rohde A. N-terminal truncated RHT-1 proteins generated by translational reinitiation cause semi-dwarfing of wheat Green Revolution alleles.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.01.002Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar) now makes possible new breeding strategies for improved GR varieties exhibiting enhanced NUE and grain yield (Figure 1B), thus reducing environmental impact. For example, Rht-B1b and Rht-D1b retain a conserved LHR1 domain (Figure 1A), which supports interaction with multiple transcription factors including GRF4 and NGR5 (Li et al., 2018Li S. Tian Y. Wu K. Ye Y. Yu J. Zhang J. Liu Q. Hu M. Li H. Tong Y. et al.Modulating plant growth-metabolism coordination for sustainable agriculture.Nature. 2018; 560: 595-600Crossref PubMed Scopus (200) Google Scholar; Wu et al., 2020Wu K. Wang S. Song W. Zhang J. Wang Y. Liu Q. Yu J. Ye Y. Li S. Chen J J. et al.Enhanced sustainable green revolution yield via nitrogen-responsive chromatin modulation in rice.Science. 2020; 367: eaaz2046Crossref PubMed Scopus (85) Google Scholar). CRISPR-Cas9 mutagenesis of LHR1 might generate mutant Rht-B1b or Rht-D1b proteins with reduced affinity for GRF4 or enhanced affinity for NGR5, thus improving NUE and/or shoot branching response, respectively. Such mutant proteins would leave grain properties unaltered, because Rht-B1b/RhtD1b is not expressed in aleurone (Figure 1B). However, LHR1 mutations might also affect interactions with growth-regulatory transcription factors, thus causing unwanted loss of yield-beneficial dwarfism. An alternative strategy might specifically mutate the LHR1-interacting regions of GRF4 or NGR5 themselves, thus reducing GRF4 or enhancing NGR5 interaction. Such mutations would enhance NUE and/or shoot branching response (specifically conferred by GRF4 or NGR5), would not reduce the dwarfism conferred by Rht-B1b/Rht-D1b, and would again leave grain properties unaltered (Figure 1B). These strategies could enable precision breeding of improved GR varieties having higher yields with reduced N fertilizer use, thus enhancing the environmental sustainability of world agriculture. This work was supported by the National Natural Science Foundation of China ( 31921005 , 91935301 , and 31830082 ), the Youth Innovation Promotion Association CAS ( 2019100 ), and the UK Biotechnology and Biological Sciences Research Council ( BB/N013611/1 and BB/S013741/1 ).