Chlorophylls and carotenoids are essential photosynthetic pigments. Plants spatiotemporally coordinate the needs of chlorophylls and carotenoids for optimal photosynthesis and fitness in response to diverse environmental and developmental cues. However, how the biosynthesis pathways of these two pigments are coordinated, particularly at posttranslational level to allow rapid control, remains largely unknown. Here, we report that the highly conserved ORANGE (OR) family proteins coordinate both pathways via posttranslationally mediating the first committed enzyme in each pathway. We demonstrate that OR family proteins physically interact with magnesium chelatase subunit I (CHLI) in chlorophyll biosynthesis pathway in addition to phytoene synthase (PSY) in carotenoid biosynthesis pathway and concurrently stabilize CHLI and PSY enzymes. We show that loss of OR genes hinders both chlorophyll and carotenoid biosynthesis, limits light-harvesting complex assembly, and impairs thylakoid grana stacking in chloroplasts. Overexpression of OR safeguards photosynthetic pigment biosynthesis and enhances thermotolerance in both Arabidopsis and tomato plants. Our findings establish a novel mechanism by which plants coordinate chlorophyll and carotenoid biosynthesis and provide a potential genetic target to generate climate-resilient crops.

Excessive accumulation of chloride (Cl-) in the aboveground tissues under saline conditions is harmful to crops. Increasing the exclusion of Cl- from shoots promotes salt tolerance in various crops. However, the underlying molecular mechanisms remain largely unknown. In this study, we demonstrated that a type A response regulator (ZmRR1) modulates Cl- exclusion from shoots and underlies natural variation of salt tolerance in maize. ZmRR1 negatively regulates cytokinin signaling and salt tolerance, likely by interacting with and inhibiting His phosphotransfer (HP) proteins that are key mediators of cytokinin signaling. A naturally occurring non-synonymous SNP variant enhances the interaction between ZmRR1 and ZmHP2, conferring maize plants with a salt-hypersensitive phenotype. We found that ZmRR1 undergoes degradation under saline conditions, leading to the release of ZmHP2 from ZmRR1 inhibition, and subsequently ZmHP2-mediated signaling improves salt tolerance primarily by promoting Cl- exclusion from shoots. Furthermore, we showed that ZmMATE29 is transcriptionally upregulated by ZmHP2-mediated signaling under highly saline conditions and encodes a tonoplast-located Cl- transporter that promotes Cl- exclusion from shoots by compartmentalizing Cl- into the vacuoles of root cortex cells. Collectively, our study provides an important mechanistic understanding of the cytokinin signaling-mediated promotion of Cl- exclusion from shoots and salt tolerance and suggests that genetic modification to promote Cl- exclusion from shoots is a promising route for developing salt-tolerant maize.

The orderly deposition of secondary cell wall (SCW) in plants is implicated in various biological programs and is precisely controlled. Although many positive and negative regulators of SCW have been documented, the molecular mechanisms underlying SCW formation coordinated with distinct cellular physiological processes during plant adaptive growth remain largely unclear. Here, we report the identification of Cellulose Synthase co-expressed Kinase1 (CSK1), which encodes a receptor-like cytoplasmic kinase, as a negative regulator of SCW formation and its signaling cascade in rice. Transcriptome deep sequencing of developing internodes and genome-wide co-expression assays revealed that CSK1 is co-expressed with cellulose synthase genes and is responsive to various stress stimuli. The increased SCW thickness and vigorous vessel transport in csk1 indicate that CSK1 functions as a negative regulator of SCW biosynthesis. Through observation of green fluorescent protein-tagged CSK1 in rice protoplasts and stable transgenic plants, we found that CSK1 is localized in the nucleus and cytoplasm adjacent to the plasma membrane. Biochemical and molecular assays demonstrated that CSK1 phosphorylates VASCULAR-RELATED NAC-DOMAIN 6 (VND6), a master SCW-associated transcription factor, in the nucleus, which reduces the transcription of a suite of SCW-related genes, thereby attenuating SCW accumulation. Consistently, genetic analyses show that CSK1 functions upstream of VND6 in regulating SCW formation. Interestingly, our physiological analyses revealed that CSK1 and VND6 are involved in abscisic acid-mediated regulation of cell growth and SCW deposition. Taken together, these results indicate that the CSK1-VND6 module is an important component of the SCW biosynthesis machinery, which coordinates SCW accumulation and adaptive growth in rice. Our study not only identifies a new regulator of SCW biosynthesis but also reveals a fine-tuned mechanism for precise control of SCW deposition, offering tools for rationally tailoring agronomic traits.

Acetylation of N4-cytidine (ac4C) has recently been discovered as a novel modification of mRNA. RNA ac4C modification has been shown to be a key regulator of RNA stability, RNA translation, and the thermal stress response. However, its existence in eukaryotic mRNAs is still controversial. In plants, the existence, distribution pattern, and potential function of RNA ac4C modification are largely unknown. Here we report the presence of ac4C in the mRNAs of both Arabidopsis thaliana and rice (Oryza sativa). By comparing two ac4C sequencing methods, we found that RNA immunoprecipitation and sequencing (acRIP-seq), but not ac4C sequencing, was suitable for plant RNA ac4C sequencing. We present transcriptome-wide atlases of RNA ac4C modification in A. thaliana and rice mRNAs obtained by acRIP-seq. Analysis of the distribution of RNA ac4C modifications showed that ac4C is enriched near translation start sites in rice mRNAs and near translation start sites and translation end sites in Arabidopsis mRNAs. The RNA ac4C modification level is positively correlated with RNA half-life and the number of splicing variants. Similar to that in mammals, the translation efficiency of ac4C target genes is significantly higher than that of other genes. Our in vitro translation results confirmed that RNA ac4C modification enhances translation efficiency. We also found that RNA ac4C modification is negatively correlated with RNA structure. These results suggest that ac4C is a conserved mRNA modification in plants that contributes to RNA stability, splicing, translation, and secondary structure formation.

Vitamin A deficiency remains a severe global health issue, which creates a need to biofortify crops with provitamin A carotenoids (PACs). Expanding plant cell capacity for synthesis and storing of PACs outside the plastids is a promising biofortification strategy that has been little explored. Here, we engineered PAC formation and sequestration in the cytosol of Nicotiana benthamiana leaves, Arabidopsis seeds, and citrus callus cells, using a fungal (Neurospora crassa) carotenoid pathway that consists of only three enzymes converting C5 isopentenyl building blocks formed from mevalonic acid into PACs, including β-carotene. This strategy led to the accumulation of significant amounts of phytoene and γ- and β-carotene, in addition to fungal, health-promoting carotenes with 13 conjugated double bonds, such as the PAC torulene, in the cytosol. Increasing the isopentenyl diphosphate pool by adding a truncated Arabidopsis hydroxymethylglutaryl-coenzyme A reductase substantially increased cytosolic carotene production. Engineered carotenes accumulate in cytosolic lipid droplets (CLDs), which represent a novel sequestering sink for storing these pigments in plant cytosol. Importantly, β-carotene accumulated in the cytosol of citrus callus cells was more light stable compared to compared with plastidial β-carotene. Moreover, engineering cytosolic carotene formation increased the number of large-sized CLDs and the levels of β-apocarotenoids, including retinal, the aldehyde corresponding to vitamin A. Collectively, our study opens up the possibility of exploiting the high-flux mevalonic acid pathway for PAC biosynthesis and enhancing carotenoid sink capacity in green and non-green plant tissues, especially in lipid-storing seeds, and thus paves the way for further optimization of carotenoid biofortification in crops.