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A transformation in plant cell wall evolution marked the emergence of grasses, grains and related species that now cover much of the globe. Their tough, less digestible cell walls arose from a new pattern of cross-linking between arabinoxylan polymers with distinctive ferulic acid residues. Despite extensive study, the biochemical mechanism of ferulic acid incorporation into cell walls remains unknown. Here we show that ferulic acid is transferred to arabinoxylans via an unexpected sucrose derivative, 3,6-O-diferuloyl sucrose (2-feruloyl-O-α-d-glucopyranosyl-(1′→2)-3,6-O-feruloyl-β-d-fructofuranoside), formed by a sucrose ferulate cycle. Sucrose gains ferulate units through sequential transfers from feruloyl-CoA, initially at the O-3 position of sucrose catalysed by a family of BAHD-type sucrose ferulic acid transferases (SFT1 to SFT4 in maize), then at the O-6 position by a feruloyl sucrose feruloyl transferase (FSFT), which creates 3,6-O-diferuloyl sucrose. An FSFT-deficient mutant of maize, disorganized wall 1(dow1), sharply decreases cell wall arabinoxylan ferulic acid content, causes accumulation of 3-O-feruloyl sucrose (α-d-glucopyranosyl-(1′→2)-3-O-feruloyl-β-d-fructofuranoside) and leads to the abortion of embryos with defective cell walls. In vivo, isotope-labelled ferulic acid residues are transferred from 3,6-O-diferuloyl sucrose onto cell wall arabinoxylans. This previously unrecognized sucrose ferulate cycle resolves a long-standing mystery surrounding the evolution of the distinctive cell wall characteristics of cereal grains, biofuel crops and related commelinid species; identifies an unexpected role for sucrose as a ferulate group carrier in cell wall biosynthesis; and reveals a new paradigm for modifying cell wall polymers through ferulic acid incorporation.

Pre-rRNA processing is essential to ribosome biosynthesis. However, the processing mechanism is not fully understood in plants. Here, we report a DEAD-box RNA helicase DEK51 that mediates the 3′ end processing of 18S and 5.8S pre-rRNA in maize (Zea maysL.). DEK51 is localized in the nucleolus, and loss of DEK51 arrests maize seed development and blocks the 3′ end processing of 18S and 5.8S pre-rRNA. DEK51 interacts with putative key factors in nuclear RNA exosome-mediated pre-rRNA processing, including ZmMTR4, ZmSMO4, ZmRRP44A, and ZmRRP6L2. This suggests that DEK51 facilitates pre-rRNA processing by interacting with the exosome. Loss of ZmMTR4 function arrests seed development and blocks the 3′ end processing of 18S and 5.8S pre-rRNA, similar to dek51. DEK51 also interacts with endonucleases ZmUTP24 and ZmRCL1, suggesting that it may also be involved in the cleavage at site A2. These results show the critical role of DEK51 in promoting 3′ end processing of pre-rRNA.

ABSTRACTPentatricopeptide repeat (PPR) proteins play an important role in post-transcriptional regulation of mitochondrial gene expression. Functions of many PPR proteins and their roles in plant growth and development remain unknown. Through characterization of an empty pericarp32(emp32) mutant, we identified the function of Emp32in mitochondrial intron splicing and seed development in maize. The loss-of-function mutant emp32shows embryo lethality with severely arrested embryo and endosperm development, and over-expression of Emp32rescues the embryo-lethality. EMP32 is a P-type PPR protein targeted to mitochondria. Loss of function in Emp32dramatically decreases the splicing efficiency of nad7intron 2, while complementation of Emp32restores the splicing efficiency. Although nad7intron 2 is partially spliced in the wild type, over-expression of Emp32does not increase the splicing efficiency. The splicing deficiency of nad7intron 2 blocks the assembly of mitochondrial complex I and dramatically reduces its activity, which may explain the embryo-lethality in emp32. In addition to the one copy of nad7in the maize mitochondrial genome, we identified one to six copies of nad7in the nuclear genomes in different maize inbred lines. These copies appear not to be expressed. Together, our results revealed that the P-type PPR protein EMP32 is required for the cis-splicing of nad7intron 2 and seed development in maize.

Pentatricopeptide repeat (PPR) proteins are a large group of eukaryote-specific RNA-binding proteins that play pivotal roles in plant organelle gene expression. Here, we report the function of PPR21 in mitochondrial intron splicing and its role in maize kernel development. PPR21 is a typical P-type PPR protein targeted to mitochondria. The ppr21mutants are arrested in embryogenesis and endosperm development, leading to embryo lethality. Null mutations of PPR21reduce the splicing efficiency of nad2intron 1, 2, and 4 and impair the assembly and activity of mitochondrial complex I. Previous studies show that the P-type PPR protein EMP12 is required for the splicing of identical introns. However, our protein interaction analyses reveal that PPR21 does not interact with EMP12. Instead, both PPR21 and EMP12 interact with the small MutS-related (SMR) domain–containing PPR protein 1 (PPR-SMR1) and the short P-type PPR protein 2 (SPR2). PPR-SMR1 interacts with SPR2, and both proteins are required for the splicing of many introns in mitochondria, including nad2intron 1, 2, and 4. These results suggest that a PPR21–(PPR-SMR1/SPR2)–EMP12 complex is involved in the splicing of nad2introns in maize mitochondria.

RNA C-to-U editing is essential for plant organellar gene expression. It is proposed that PPR-E+ proteins bind to target sites and recruit cytidine deaminase AtDYW2 facilitated by AtNUWA. We analyzed the function of ZmNUWA, ZmDYW2A, and ZmDYW2B and their relationship with other editing factors in maize. The zmdyw2aand zmdyw2bsingle mutants are normal, but the zmdyw2a::zmdyw2band zmnuwamutants are severely arrested in seed development. ZmNUWA, ZmDYW2A, and ZmDYW2B are dual-localized in mitochondria and plastids. Loss of ZmNUWA decreases the editing at 99 mitochondrial sites and 8 plastid sites. Surprisingly, loss of ZmDYW2A:ZmDYW2B affects almost the same set of sites targeted by PPR-E+ proteins. ZmNUWA interacts with ZmDYW2A and ZmDYW2B, suggesting that ZmNUWA recruits ZmDYW2A/2B in the editing of PPR-E+ protein targeted sites in maize. Further protein interaction analyses show that ZmNUWA and ZmDYW2A/2B interact with ZmMORF1, ZmMORF8, ZmMORF2, and ZmMORF9; and ZmOZ1 interacts with ZmORRM1, ZmDYW2A, ZmDYW2B, ZmMORF8, and ZmMORF9. These results suggest that the mitochondrial PPR-E+ editosome contains PPR-E+, ZmDYW2A/2B, ZmNUWA, and ZmMORF1/8, whereas the plastid PPR-E+ editosome composes of PPR-E+, ZmDYW2A/2B, ZmNUWA, ZmMORF2/8/9, ZmORRM1 and ZmOZ1 in maize.

Gibberellin (GA) plays important roles through plant growth and development. However, where GA is synthesized inside a cell and how it regulates sex determination is obscure. We analyzed the classic dwarf1(d1) mutant in maize and revealed that D1 encodes GA 3-oxidase converting inactive GA intermediates to bioactive GA. As such, the D1 protein marks the sites where GA is potentially synthesized. Interestingly, the D1 protein was found to localize in the cytosol and nucleus, a dual-localization coinciding with the GA receptor. The same result was found for GA 20-oxidase catalyzing the upstream reaction. These results suggest that GA can be synthesized in the cytosol and nucleus. The D1 protein was highly and specifically expressed in the stamen primordia in the ear florets, but low in the whole tassel. Hence it is possible that low level of GA in the tassel is insufficient to suppress stamen development. As jasmonic acid (JA) plays antagonistic role to GA in the tassel florets, here we propose a model to explain this antagonism effect on the regulation of the stamen and pistil organ development in the tassel florets in maize.