An alanine to valine mutation of glutamyl-tRNA reductase enhances 5-aminolevulinic acid synthesis in rice.

KEY MESSAGE: An alanine to valine mutation of glutamyl-tRNA reductase's 510th amino acid improves 5-aminolevulinic acid synthesis in rice. 5-aminolevulinic acid (ALA) is the common precursor of all tetrapyrroles and plays an important role in plant growth regulation. ALA is synthesized from glutamate, catalyzed by glutamyl-tRNA synthetase (GluRS), glutamyl-tRNA reductase (GluTR), and glutamate-1-semialdehyde aminotransferase (GSAT). In Arabidopsis, ALA synthesis is the rate-limiting step in tetrapyrrole production via GluTR post-translational regulations. In rice, mutations of GluTR and GSAT homologs are known to confer chlorophyll deficiency phenotypes; however, the enzymatic activity of rice GluRS, GluTR, and GSAT and the post-translational regulation of rice GluTR have not been investigated experimentally. We have demonstrated that a suppressor mutation in rice partially reverts the xantha trait. In the present study, we first determine that the suppressor mutation results from a G → A nucleotide substitution of OsGluTR (and an A → V change of its 510th amino acid). Protein homology modeling and molecular docking show that the OsGluTRA510V mutation increases its substrate binding. We then demonstrate that the OsGluTRA510V mutation increases ALA synthesis in Escherichia coli without affecting its interaction with OsFLU. We further explore homologous genes encoding GluTR across 193 plant species and find that the amino acid (A) is 100% conserved at the position, suggesting its critical role in GluTR. Thus, we demonstrate that the gain-of-function OsGluTRA510V mutation underlies suppression of the xantha trait, experimentally proves the enzymatic activity of rice GluRS, GluTR, and GSAT in ALA synthesis, and uncovers conservation of the alanine corresponding to the 510th amino acid of OsGluTR across plant species.

Physiological mechanisms underlying OsNAC5-dependent tolerance of rice plants to abiotic stress.

To understand the functions of transcription factor OsNAC5 in response to abiotic stress, we generated transgenic rice plants with knockdown OsNAC5 by RNA-interfered (RNAi) and overexpressing OsNAC5, and investigated the effects of cold, drought and salt stress on wild-type (WT), RNAi and overexpression rice lines. Our results demonstrated that RNAi lines became less tolerant to these stresses than WT plants, while overexpression of OsNAC5 in Arabidopsis and rice enhanced tolerance to these stresses. The mechanisms underlying the changes in tolerance of the transgenic rice plants to abiotic stresses were explored by measuring free proline (Pro) and soluble sugar contents in WT and transgenic plants. Accumulation of Pro and soluble sugars was positively correlated with OsNAC5 expression levels. The less accumulation of Pro in RNAi lines may be accounted for by inhibition of Pro synthesis and transport at transcriptional levels. In addition, knockdown and overexpression of OsNAC5 enhanced and reduced accumulation of malondialdehyde and H(2)O(2), suggesting that knockdown of OsNAC5 renders RNAi plants more susceptible to oxidative damage. The RNAi lines displayed higher Na(+)/K(+) ratio due to greater accumulation of Na(+) ions than WT under salt stress conditions, and expression of genes encoding tonoplast Na(+)/H(+) antiporter was lower in RNAi lines than in WT under both control and salt-stressed conditions. Seed germination of RNAi and overexpression plants was more and less inhibited by salt and mannitol than that of WT, respectively. Seed germination of overexpression and RNAi plants was more and less sensitive than that of WT to ABA. These findings highlight the important role of OsNAC5 played in the tolerance of rice plants to abiotic stress by regulating downstream targets associated with accumulation of compatible solutes, Na(+) ions, H(2)O(2) and malondialdehyde.

Identification, Characterization, and Mutational Analysis of a Probable KEAP1 Ortholog in Rice (Oryza sativa L.).

The Kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor E2-related factor 2 (NRF2) module is a key component in the detoxification and antioxidant system in animals, which plays crucial roles in cell homeostasis and cytoprotection, and consequently in carcinogenesis and disease development. However, this system seems to have diverged throughout evolution across different organisms, and the question of whether a similar system exists in plants has thus far remained unresolved. In this study, a KEAP1 ortholog was identified in rice (Oryza sativa L., OsKEAP1) and its properties were characterized via in silico and laboratory studies. To reveal OsKEAP1's function, two knockdown mutants, oskeap1-1 and oskeap1-2, were generated by targeted mutagenesis in the 5' untranslated region (UTR) using the CRISPR-Cas9 system. In silico analysis showed that OsKEAP1 has a Kelch-repeat domain which is identical to those of animals and a plant-specific development and cell death (DCD) domain in place of the broad-complex, tramtrack, bric-a-brac (BTB) domain found in animals. Orthologs of OsKEAP1 are present across plant species and all have the DCD domain and the Kelch-repeat domain. OsKEAP1 was proven to be localized to both the cytoplasm and nucleus, in contrast to the exclusive cytoplasm localization of animal KEAP1. Single nucleotide insertions in the 5' UTR significantly reduced the transcription level of OsKEAP1 in the oskeap1-1 and oskeap1-2 mutants. The oskeap1 mutations greatly impaired plant growth and development, resulting in significant declines in a majority of agronomic and yield-related traits, i.e., plant height, panicle length, grain number per plant, and seed-set rate. The downregulation of OsKEAP1 increased the levels of H2O2, malondialdehyde, and proline while significantly decreasing the expression of two catalase genes in seedlings grown under normal and salt-stressed conditions. The changes in the above phenotypes are either positively or negatively correlated with the degree of OsKEAP1 downregulation. Altogether, we identified a probable KEAP1 ortholog in rice, revealed its unique subcellular localization, and demonstrated its important functions in vegetative and reproductive growth via regulation of the antioxidant response in plants.

Gene editing: an instrument for practical application of gene biology to plant breeding.

Plant breeding is well recognized as one of the most important means to meet food security challenges caused by the ever-increasing world population. During the past three decades, plant breeding has been empowered by both new knowledge on trait development and regulation (e.g., functional genomics) and new technologies (e.g., biotechnologies and phenomics). Gene editing, particularly by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) and its variants, has become a powerful technology in plant research and may become a game-changer in plant breeding. Traits are conferred by coding and non-coding genes. From this perspective, we propose different editing strategies for these two types of genes. The activity of an encoded enzyme and its quantity are regulated at transcriptional and post-transcriptional, as well as translational and post-translational, levels. Different strategies are proposed to intervene to generate gene functional variations and consequently phenotype changes. For non-coding genes, trait modification could be achieved by regulating transcription of their own or target genes via gene editing. Also included is a scheme of protoplast editing to make gene editing more applicable in plant breeding. In summary, this review provides breeders with a host of options to translate gene biology into practical breeding strategies, i.e., to use gene editing as a mechanism to commercialize gene biology in plant breeding.