Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice.

How reciprocal regulation of carbon and nitrogen metabolism works is a long-standing question. In plants, glucose and nitrate are proposed to act as signaling molecules, regulating carbon and nitrogen metabolism via largely unknown mechanisms. Here, we show that the MYB-related transcription factor ARE4 coordinates glucose signaling and nitrogen utilization in rice. ARE4 is retained in the cytosol in complexing with the glucose sensor OsHXK7. Upon sensing a glucose signal, ARE4 is released, is translocated into the nucleus, and activates the expression of a subset of high-affinity nitrate transporter genes, thereby boosting nitrate uptake and accumulation. This regulatory scheme displays a diurnal pattern in response to circadian changes of soluble sugars. The are4 mutations compromise in nitrate utilization and plant growth, whereas overexpression of ARE4 increases grain size. We propose that the OsHXK7-ARE4 complex links glucose to the transcriptional regulation of nitrogen utilization, thereby coordinating carbon and nitrogen metabolism.

SEMI-ROLLED LEAF 10 stabilizes catalase isozyme B to regulate leaf morphology and thermotolerance in rice (Oryza sativa L.).

Plant architecture and stress tolerance play important roles in rice breeding. Specific leaf morphologies and ideal plant architecture can effectively improve both abiotic stress resistance and rice grain yield. However, the mechanism by which plants simultaneously regulate leaf morphogenesis and stress resistance remains elusive. Here, we report that SRL10, which encodes a double-stranded RNA-binding protein, regulates leaf morphology and thermotolerance in rice through alteration of microRNA biogenesis. The srl10 mutant had a semi-rolled leaf phenotype and elevated sensitivity to high temperature. SRL10 directly interacted with catalase isozyme B (CATB), and the two proteins mutually increased one other's stability to enhance hydrogen peroxide (H2 O2 ) scavenging, thereby contributing to thermotolerance. The natural Hap3 (AGC) type of SRL10 allele was found to be present in the majority of aus rice accessions, and was identified as a thermotolerant allele under high temperature stress in both the field and the growth chamber. Moreover, the seed-setting rate was 3.19 times higher and grain yield per plant was 1.68 times higher in near-isogenic line (NIL) carrying Hap3 allele compared to plants carrying Hap1 allele under heat stress. Collectively, these results reveal a new locus of interest and define a novel SRL10-CATB based regulatory mechanism for developing cultivars with high temperature tolerance and stable yield. Furthermore, our findings provide a theoretical basis for simultaneous breeding for plant architecture and stress resistance.

LEAF TIP RUMPLED 1 Regulates Leaf Morphology and Salt Tolerance in Rice.

Leaf morphology is one of the important traits related to ideal plant architecture and is an important factor determining rice stress resistance, which directly affects yield. Wax layers form a barrier to protect plants from different environmental stresses. However, the regulatory effect of wax synthesis genes on leaf morphology and salt tolerance is not well-understood. In this study, we identified a rice mutant, leaf tip rumpled 1 (ltr1), in a mutant library of the classic japonica variety Nipponbare. Phenotypic investigation of NPB and ltr1 suggested that ltr1 showed rumpled leaf with uneven distribution of bulliform cells and sclerenchyma cells, and disordered vascular bundles. A decrease in seed-setting rate in ltr1 led to decreased per-plant grain yield. Moreover, ltr1 was sensitive to salt stress, and LTR1 was strongly induced by salt stress. Map-based cloning of LTR1 showed that there was a 2-bp deletion in the eighth exon of LOC_Os02g40784 in ltr1, resulting in a frameshift mutation and early termination of transcription. Subsequently, the candidate gene was confirmed using complementation, overexpression, and knockout analysis of LOC_Os02g40784. Functional analysis of LTR1 showed that it was a wax synthesis gene and constitutively expressed in entire tissues with higher relative expression level in leaves and panicles. Moreover, overexpression of LTR1 enhanced yield in rice and LTR1 positively regulates salt stress by affecting water and ion homeostasis. These results lay a theoretical foundation for exploring the molecular mechanism of leaf morphogenesis and stress response, providing a new potential strategy for stress-tolerance breeding.

Regulation of nitrogen starvation responses by the alarmone (p)ppGpp in rice.

Nitrogen is an essential macronutrient for all living organisms and is critical for crop productivity and quality. In higher plants, inorganic nitrogen is absorbed through roots and then assimilated into amino acids by the highly conserved glutamine synthetase/glutamine:2-oxoglutarate aminotransferase (GS/GOGAT) cycle. How nitrogen metabolism and nitrogen starvation responses of plants are regulated remains largely unknown. Previous studies revealed that mutations in the rice ABNORMAL CYTOKININ RESPONSE1 (ABC1) gene encoding Fd-GOGAT cause a typical nitrogen deficiency syndrome. Here, we show that ARE2 (for ABC1 REPRESSOR2) is a key regulator of nitrogen starvation responses in rice. The are2 mutations partially rescue the nitrogen-deficient phenotype of abc1 and the are2 mutants show enhanced tolerance to nitrogen deficiency, suggesting that ARE2 genetically interacts with ABC1/Fd-GOGAT. ARE2 encodes a chloroplast-localized RelA/SpoT homolog protein that catalyzes the hydrolysis of guanosine pentaphosphate or tetraphosphate (p)ppGpp, an alarmone regulating the stringent response in bacteria under nutritional stress conditions. The are2 mutants accumulate excessive amounts of (p)ppGpp, which correlate with lower levels of photosynthetic proteins and higher amino acid levels. Collectively, these observations suggest that the alarmone (p)ppGpp mediates nitrogen stress responses and may constitute a highly conserved mechanism from bacteria to plants.

The Ghd7 transcription factor represses ARE1 expression to enhance nitrogen utilization and grain yield in rice.

The genetic improvement of nitrogen use efficiency (NUE) of crops is vital for grain productivity and sustainable agriculture. However, the regulatory mechanism of NUE remains largely elusive. Here, we report that the rice Grain number, plant height, and heading date7 (Ghd7) gene genetically acts upstream of ABC1 REPRESSOR1 (ARE1), a negative regulator of NUE, to positively regulate nitrogen utilization. As a transcriptional repressor, Ghd7 directly binds to two Evening Element-like motifs in the promoter and intron 1 of ARE1, likely in a cooperative manner, to repress its expression. Ghd7 and ARE1 display diurnal expression patterns in an inverse oscillation manner, mirroring a regulatory scheme based on these two loci. Analysis of a panel of 2656 rice varieties suggests that the elite alleles of Ghd7 and ARE1 have undergone diversifying selection during breeding. Moreover, the allelic distribution of Ghd7 and ARE1 is associated with the soil nitrogen deposition rate in East Asia and South Asia. Remarkably, the combination of the Ghd7 and ARE1 elite alleles substantially improves NUE and yield performance under nitrogen-limiting conditions. Collectively, these results define a Ghd7-ARE1-based regulatory mechanism of nitrogen utilization, providing useful targets for genetic improvement of rice NUE.

PHOTO-SENSITIVE LEAF ROLLING 1 encodes a polygalacturonase that modifies cell wall structure and drought tolerance in rice.

The biosynthesis and modification of cell wall composition and structure are controlled by hundreds of enzymes and have a direct consequence on plant growth and development. However, the majority of these enzymes has not been functionally characterised. Rice mutants with leaf-rolling phenotypes were screened in a field. Phenotypic analysis under controlled conditions was performed for the selected mutant and the relevant gene was identified by map-based cloning. Cell wall composition was analysed by glycome profiling assay. We identified a photo-sensitive leaf rolling 1 (psl1) mutant with 'napping' (midday depression of photosynthesis) phenotype and reduced growth. The PSL1 gene encodes a cell wall-localised polygalacturonase (PG), a pectin-degrading enzyme. psl1 with a 260-bp deletion in its gene displayed leaf rolling in response to high light intensity and/or low humidity. Biochemical assays revealed PG activity of recombinant PSL1 protein. Significant modifications to cell wall composition in the psl1 mutant compared with the wild-type plants were identified. Such modifications enhanced drought tolerance of the mutant plants by reducing water loss under osmotic stress and drought conditions. Taken together, PSL1 functions as a PG that modifies cell wall biosynthesis, plant development and drought tolerance in rice.

Genetic variations in ARE1 mediate grain yield by modulating nitrogen utilization in rice.

In crops, nitrogen directly determines productivity and biomass. However, the improvement of nitrogen utilization efficiency (NUE) is still a major challenge in modern agriculture. Here, we report the characterization of are1, a genetic suppressor of a rice fd-gogat mutant defective in nitrogen assimilation. ARE1 is a highly conserved gene, encoding a chloroplast-localized protein. Loss-of-function mutations in ARE1 cause delayed senescence and result in 10-20% grain yield increases, hence enhance NUE under nitrogen-limiting conditions. Analysis of a panel of 2155 rice varieties reveals that 18% indica and 48% aus accessions carry small insertions in the ARE1 promoter, which result in a reduction in ARE1 expression and an increase in grain yield under nitrogen-limiting conditions. We propose that ARE1 is a key mediator of NUE and represents a promising target for breeding high-yield cultivars under nitrogen-limiting condition.

Rice Ferredoxin-Dependent Glutamate Synthase Regulates Nitrogen-Carbon Metabolomes and Is Genetically Differentiated between japonica and indica Subspecies.

Plants assimilate inorganic nitrogen absorbed from soil into organic forms as Gln and Glu through the glutamine synthetase/glutamine:2-oxoglutarate amidotransferase (GS/GOGAT) cycle. Whereas GS catalyzes the formation of Gln from Glu and ammonia, GOGAT catalyzes the transfer of an amide group from Gln to 2-oxoglutarate to produce two molecules of Glu. However, the regulatory role of the GS/GOGAT cycle in the carbon-nitrogen balance is not well understood. Here, we report the functional characterization of rice ABNORMAL CYTOKININ RESPONSE 1 (ABC1) gene that encodes a ferredoxin-dependent (Fd)-GOGAT. The weak mutant allele abc1-1 mutant shows a typical nitrogen-deficient syndrome, whereas the T-DNA insertional mutant abc1-2 is seedling lethal. Metabolomics analysis revealed the accumulation of an excessive amount of amino acids with high N/C ratio (Gln and Asn) and several intermediates in the tricarboxylic acid cycle in abc1-1, suggesting that ABC1 plays a critical role in nitrogen assimilation and carbon-nitrogen balance. Five non-synonymous single-nucleotide polymorphisms were identified in the ABC1 coding region and characterized as three distinct haplotypes, which have been highly and specifically differentiated between japonica and indica subspecies. Collectively, these results suggest that ABC1/OsFd-GOGAT is essential for plant growth and development by modulating nitrogen assimilation and the carbon-nitrogen balance.

Rice TUTOU1 Encodes a Suppressor of cAMP Receptor-Like Protein That Is Important for Actin Organization and Panicle Development.

Panicle development, a key event in rice (Oryza sativa) reproduction and a critical determinant of grain yield, forms a branched structure containing multiple spikelets. Genetic and environmental factors can perturb panicle development, causing panicles to degenerate and producing characteristic whitish, small spikelets with severely reduced fertility and yield; however, little is known about the molecular basis of the formation of degenerating panicles in rice. Here, we report the identification and characterization of the rice panicle degenerative mutant tutou1 (tut1), which shows severe defects in panicle development. The tut1 also shows a pleiotropic phenotype, characterized by short roots, reduced plant height, and abnormal development of anthers and pollen grains. Molecular genetic studies revealed that TUT1 encodes a suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein family verprolin-homologous (SCAR/WAVE)-like protein. We found that TUT1 contains conserved functional domains found in eukaryotic SCAR/WAVE proteins, and was able to activate Actin-related protein2/3 to promote actin nucleation and polymerization in vitro. Consistently, tut1 mutants show defects in the arrangement of actin filaments in trichome. These results indicate that TUT1 is a functional SCAR/WAVE protein and plays an important role in panicle development.

Peptidyl-prolyl isomerization targets rice Aux/IAAs for proteasomal degradation during auxin signalling.

In plants, auxin signalling is initiated by the auxin-promoted interaction between the auxin receptor TIR1, an E3 ubiquitin ligase, and the Aux/IAA transcriptional repressors, which are subsequently degraded by the proteasome. Gain-of-function mutations in the highly conserved domain II of Aux/IAAs abolish the TIR1-Aux/IAA interaction and thus cause an auxin-resistant phenotype. Here we show that peptidyl-prolyl isomerization of rice OsIAA11 catalysed by LATERAL ROOTLESS2 (LRT2), a cyclophilin-type peptidyl-prolyl cis/trans isomerase, directly regulates the stability of OsIAA11. NMR spectroscopy reveals that LRT2 efficiently catalyses the cis/trans isomerization of OsIAA11. The lrt2 mutation reduces OsTIR1-OsIAA11 interaction and consequently causes the accumulation of a higher level of OsIAA11 protein. Moreover, knockdown of the OsIAA11 expression partially rescues the lrt2 mutant phenotype in lateral root development. Together, these results illustrate cyclophilin-catalysed peptidyl-prolyl isomerization promotes Aux/IAA degradation, as a mechanism regulating auxin signalling.