Modulating plant growth-metabolism coordination for sustainable agriculture

Modulating plant growth-metabolism coordination for sustainable agriculture

Enhancing global food security by increasing the productivity of green revolution varieties of cereals risks increasing the collateral environmental damage produced by inorganic nitrogen fertilizers. Improvements in the efficiency of nitrogen use of crops are therefore essential; however, they require an in-depth understanding of the co-regulatory mechanisms that integrate growth, nitrogen assimilation and carbon fixation. Here we show that the balanced opposing activities and physical interactions of the rice GROWTH-REGULATING FACTOR 4 (GRF4) transcription factor and the growth inhibitor DELLA confer homeostatic co-regulation of growth and the metabolism of carbon and nitrogen. GRF4 promotes and integrates nitrogen assimilation, carbon fixation and growth, whereas DELLA inhibits these processes. As a consequence, the accumulation of DELLA that is characteristic of green revolution varieties confers not only yield-enhancing dwarfism, but also reduces the efficiency of nitrogen use. However, the nitrogen-use efficiency of green revolution varieties and grain yield are increased by tipping the GRF4-DELLA balance towards increased GRF4 abundance. Modulation of plant growth and metabolic co-regulation thus enables novel breeding strategies for future sustainable food security and a new green revolution.

The green revolution of the 1960s boosted crop yields, and was partly driven by widespread adoption of semi-dwarf green revolution varieties of cereals (GRVs)1-4. GRV semi-dwarfism is due to the accumulation of growth-repressing DELLA proteins (DELLAs) conferred by mutant alleles at the Rht (wheat)5,6 and SD1 (rice)7,8 loci. In normal plants, gibberellin (GA) promotes growth by stimulating the destruction of DELLAs9,10. Mutant wheat GRV DELLAs5 are resistant to GA-stimulated destruction, whereas the rice GRV mutant sdl allele reduces bioactive GA abundance11,12, thus increasing accumulation of the DELLA protein SLR1 (Fig. la, b). The conferred semi-dwarfism causes GRV resistance to yield-reducing 'lodging' (flattening of plants by wind and rain)4.

GRV lodging resistance is enhanced by relative insensitivity to nitro- gen. For example, the nitrogen-induced increase in Nanjing6 (NJ6) plant height is reduced in NJ6-sd1 (Fig. 1c), and the Rht-B1b GRV allele confers similar properties on wheat (Fig. 1d). Although DELLA accu- mulation inhibits GRV growth nitrogen response, nitrogen allocation to grain continues, thus combining enhanced harvestable yield with reduced lodging risk from increased nitrogen supply1,4,5,7,8. These prop- cultivation over the past 50 years3, <LATEX>\begin{array}{} { \text { erties drove the raple sption of semi-dwarfing alleles in current ellif } } \\ { \text { and also ensured retention of } s e m i - d w a r \text { with reduced nitrogen-us } } \end{array}</LATEX> varieties5,6,12. However, GRVs efficiency (NUE)13. Accordingly, mutant sd1 and Rht alleles inhibit nitrogen uptake. For example, ammonium <LATEX>\left( \mathrm { N H } _ { 4 } ^ { + } \right)</LATEX> is the majority nitro- gen source for anaerobic paddy-field rice roots14. Although <LATEX>\mathrm { N J 6 } ^ { { } ^ { 1 5 } \mathrm { N H } _ { 4 } ^ { + } }</LATEX> uptake is regulated by nitrogen (the uptake rate is reduced by increasing nitrogen supply), sd1 reduces the underlying NJ6-sd1 uptake rate, and also interferes with its nitrogen-responsive regulation (Fig. 1e). Similarly, with nitrate (NO3) being the majority nitrogen source in aerobic soils15, the mutant Rht-B1b allele affects both underlying and nitrogen-regulated 15NO3 uptake in wheat (Fig. 1f). Thus, DELLA accumulation confers combined semi-dwarfism, reduced growth nitro- gen response and reduced nitrogen uptake to GRVs. In consequence, achievement of high GRV yield requires environmentally damaging https://doi.org/10.1038/s41586-018-0415-5

nitrogen fertilizer inputs16. Development of new GRVs that combine high yields with reduced nitrogen supply is thus an urgent goal for global sustainable agriculture2,17. We therefore analysed GRV growth- metabolism integration, reasoning that our discoveries might in turn enable development of new GRVs with improved NUE.

GRF4 promotes rice GRV ammonium uptake

We found approximately threefold variation in the <LATEX>{ } ^ { 1 5 } \mathrm { N H } _ { 4 } ^ { + }</LATEX> uptake rates of 36 sd1-containing indica rice varieties and the SD1-containing NJ6 control (Fig. 2a), then crossed NM73 (having the highest rate; Fig. 2a) with NJ6 (recurrent parent) to generate a BC1F2 population. Quantitative trait locus (QTL) analysis of <LATEX>{ } ^ { 1 5 } \mathrm { N H } _ { 4 } ^ { + }</LATEX> uptake rates revealed two logarithm of odds (LOD)-score peaks (quantitative trait loci NGR1 and NGR2 (qNGR1 and qNGR2), Fig. 2b; Supplementary Table 1). Although the NM73 qngr1 allele coincides in map position with <LATEX>s d 1 ^ { 7 , 8 } ,</LATEX> the molecular identity of the NM73 qngr2 allele, which was associated with increased <LATEX>{ } ^ { 1 5 } \mathrm { N H } _ { 4 } ^ { \dagger }</LATEX> uptake rates, was unknown. Positional mapping localized qngr2 to GRF418-20 (Extended Data Fig. la), suggesting a pre- viously unknown function in <LATEX>\mathrm { N H } _ { A } ^ { + }</LATEX> uptake regulation. Because a NM73 (GRF4ngr2) allele heterozygote has a higher rate than a NJ6 (GRF4NGR2) allele homozygote (Extended Data Fig. 1b), GRF4"gr2 semi-dominantly increases <LATEX>\mathrm { N H } _ { 4 } ^ { + }</LATEX> uptakes. An NJ6-GRF4ngr2 isogenic line accordingly exhibited increased NH4 uptake rates (versus NJ6; Fig. 2c), and increased GRF4 mRNA and GRF4 protein abundances (Fig. 2d, Extended Data Fig. 1c). Furthermore, RNA interference targeting GRF4 reduced the high 15NH & uptake rate of NJ6-GRF4ngr2, whereas trans- genic expression of GRF4ngr2 mRNA from its native promoter increased 15NH + uptake (Fig. 2c, Extended Data Fig. 1c).Overexpression of either GRF4NGR2 or GRF4ngr2 mRNA from the constitutive rice Actin1 pro- moter conferred increased 15NH4 uptake rates to NJ6 (Fig. 2c, Extended Data Fig. 1c). Thus, GRF4"gr2 is equivalent to qngr2, confers an increased 15NH & uptake rate to NM73 and counteracts the repres- sive effects of sd1-mediated SLR1 accumulation.

Fig. 1 | DELLA accumulation inhibits growth, nitrogen response and nitrogen uptake of rice and wheat GRVs. a, Indica rice variety NJ6 and near-isogenic NJ6-sd1 plants. Scale bar, 15 cm. b, Accumulation of SLR1. Heat shock protein 90 (HSP90) serves as loading control. Blots are

<LATEX>\begin{array}{} { \text { representative of three experiments performed independently with } } \\ { \text { similar results. } c , d , \text { Heights of rice \left(c\right) and wheat } \left( d \right) \text { plants. Data ar } } \end{array}</LATEX> <LATEX>\begin{array}{} { \text { mean } \pm \text { s.e.m. } \left( n = 3 0 \right) . e , { } ^ { 1 5 } N H _ { 4 } ^ { + } \text { upte rake rates num nurg nurogen supp } } \\ { \left( 0 . 1 5 N , 0 . 1 8 7 5 m M N H _ { 4 } N O _ { 3 } ; 0 . 3 N , 0 . 3 7 5 m M N H _ { 4 } N O _ { 3 } ; 0 . 6 N , 0 . 7 5 m N \right) } \end{array}</LATEX> NH4NO3; 1N, 1.25 mM NH4NO3). <LATEX>f , ^ { 1 5 } N O _ { 3 } ^ { - }</LATEX> uptake nitrogen supply (0.15N, 0.1875 mM Ca(NO3)2; 0.3N, <LATEX>\sin \mathrm { v a r y i n g }</LATEX> 0.6N, 0.75 mM Ca(NO3)2; 1N, 1.25 mM Ca(NO3)2). Data in e, f are mean <LATEX>\pm s . e . m .</LATEX> <LATEX>\left( n = 9 \right) .</LATEX> c-f, Different letters denote significant differences <LATEX>\left( P < 0 . 0 5 \right)</LATEX> from a Duncan's multiple range test.

GRF4NGR2 (NJ6) and GRF4ngr2 (NM73) allelic comparisons revealed multiple single nucleotide polymorphisms (SNPs) (Extended Data Fig. 1a, d), two of which (g.1187T>A and <LATEX>g</LATEX> g.1188C>A in exon 3)

Fig. 2 | GRF4 regulates rice <LATEX>\mathrm { N H } _ { 4 } ^ { + }</LATEX> uptake and growth response to nitrogen availability. a, Variation in <LATEX>{ } ^ { 1 5 } \mathrm { N H } _ { 4 } ^ { + }</LATEX> uptake and grain yield. Four- week-old rice plants ("NH} uptake assays) were grown hydroponically with high nitrogen supply (1.25 mM NH4NO3). Field-grown rice plants (yield assays) were grown with urea supply (210 kg ha-1). Data are mean ± s.e.m. of six plots (each plot contained 220 plants) per line. b, QTL analysis. c, 15NH uptake rates. d, Accumulation of GRF4. e, GRF4 transcript abundance in NJ6 roots grown in increasing nitrogen supply (0.15N, 0.1875 mM NH4NO3; 0.3N, 0.375 mM NH4NO3; 0.6N, 0.75 mM NH4NO3; 1N, 1.25 mM NH4NO3). Transcription is measured relative to