Role of SMU Homologues in Pre-mRNA Splicing During Maize and Arabidopsis Development

A maize (Zea mays ssp. mays) opaque mutant, mto38 (Mutator-taggedopaque 38), was shown to cosegregate with a Mutator-tagged genomicfragment. Sequence analysis of the DNA indicated that it contained a genesimilar to the smu-2 (suppressor of mec-8 unc-52) gene in nematodes. Previous studies showed that the mutations in either thenematode smu-1 or smu-2 genes affect splicing of the unc-52 pre-mRNA, and SMU-1 protein interacts with SMU-2 protein. In addition, human homologues of SMU-1 and SMU-2 proteins wereidentified from human spliceosome. Thus, animal SMU-1 and SMU-2 homologuesappear to play a role in pre-mRNA splicing. Plant SMU-1 and SMU-2 homologueshave not been characterized. This study demonstrated that a Mutator insertion in Zmsmu2 (Zea mays homologue of nematode smu-2) geneis responsible for multiple mutant phenotypes. Transcript profiling of mto38/zmsmu2-1endosperm revealed that defective rRNA processing and inefficient proteinsynthesis in the mutant can explain the mutant endosperm phenotypes.Furthermore, splicing of multiple pre-mRNAs is altered in zmsmu2-1endosperm, indicating a regulatory role for ZmSMU2 in pre-mRNA splicing. Thisstudy also describes the AtSMU1 and AtSMU2 genes, which encodethe Arabidopsis homologues of nematode SMU-1 and SMU-2, respectively.The SMU-2 homologues of Arabidopsis and maize physically interact with their corresponding SMU-1 homologues. Genetic analysis indicated that the AtSMU1 and AtSMU2 genes are in the same genetic pathway, and mutations in AtSMU1 and AtSMU2 also result in altered splicing of pre-mRNAs, as was true for zmsmu2. Taken together, the data presented in this study indicate a rolefor plant SMU-2 homologues in pre-mRNA splicing

The ATG Autophagic Conjugation System in Maize: ATG Transcripts and Abundance of the ATG8-Lipid Adduct Are Regulated by Development and Nutrient Availability1[W][OA]

Plants employ sophisticated mechanisms to recycle intracellular constituents needed for growth, development, and survival under nutrient-limiting conditions. Autophagy is one important route in which cytoplasm and organelles are sequestered in bulk into vesicles and subsequently delivered to the vacuole for breakdown by resident hydrolases. The formation and trafficking of autophagic vesicles are directed in part by associated conjugation cascades that couple the AUTOPHAGY-RELATED8 (ATG8) and ATG12 proteins to their respective targets, phosphatidylethanolamine and the ATG5 protein. To help understand the importance of autophagy to nutrient remobilization in cereals, we describe here the ATG8/12 conjugation cascades in maize (Zea mays) and examine their dynamics during development, leaf senescence, and nitrogen and fixed-carbon starvation. From searches of the maize genomic sequence using Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) counterparts as queries, we identified orthologous loci encoding all components necessary for ATG8/12 conjugation, including a five-member gene family expressing ATG8. Alternative splicing was evident for almost all Atg transcripts, which could have important regulatory consequences. In addition to free ATG8, its membrane-associated, lipidated form was detected in many maize tissues, suggesting that its conjugation cascade is active throughout the plant at most, if not all, developmental stages. Levels of Atg transcripts and/or the ATG8-phosphatidylethanolamine adduct increase during leaf senescence and nitrogen and fixed-carbon limitations, indicating that autophagy plays a key role in nutrient remobilization. The description of the maize ATG system now provides a battery of molecular and biochemical tools to study autophagy in this crop under field conditions

Plant SMU-1 and SMU-2 Homologues Regulate Pre-mRNA Splicing and Multiple Aspects of Development1[C][W][OA]

In eukaryotes, alternative splicing of pre-mRNAs contributes significantly to the proper expression of the genome. However, the functions of many auxiliary spliceosomal proteins are still unknown. Here, we functionally characterized plant homologues of nematode suppressors of mec-8 and unc-52 (smu). We compared transcript profiles of maize (Zea mays) smu2 endosperm with those of wild-type plants and identified pre-mRNA splicing events that depend on the maize SMU2 protein. Consistent with a conserved role of plant SMU-2 homologues, Arabidopsis (Arabidopsis thaliana) smu2 mutants also show altered splicing of similar target pre-mRNAs. The Atsmu2 mutants occasionally show developmental phenotypes, including abnormal cotyledon numbers and higher seed weights. We identified AtSMU1 as one of the SMU2-interacting proteins, and Atsmu1 mutations cause similar developmental phenotypes with higher penetrance than Atsmu2. The AtSMU2 and AtSMU1 proteins are localized to the nucleus and highly prevalent in actively dividing tissues. Taken together, our data indicated that the plant SMU-1 and SMU-2 homologues appear to be involved in splicing of specific pre-mRNAs that affect multiple aspects of development

The phosphatidylinositol 3-phosphate effector FYVE3 regulates FYVE2-dependent autophagy in Arabidopsis thaliana

Phosphatidylinositol 3-phosphate (PI3P) is a signaling phospholipid that play a key role in endomembrane trafficking, specifically autophagy and endosomal trafficking. However, the mechanisms underlying the contribution of PI3P downstream effectors to plant autophagy remain unknown. Known PI3P effectors for autophagy in Arabidopsis thaliana include ATG18A (Autophagy-related 18A) and FYVE2 (Fab1p, YOTB, Vac1p, and EEA1 2), which are implicated in autophagosome biogenesis. Here, we report that FYVE3, a paralog of plant-specific FYVE2, plays a role in FYVE2-dependent autophagy. Using yeast two-hybrid and bimolecular fluorescence complementation assays, we determined that the FYVE3 protein was associated with autophagic machinery containing ATG18A and FYVE2, by interacting with ATG8 isoforms. The FYVE3 protein was transported to the vacuole, and the vacuolar delivery of FYVE3 relies on PI3P biosynthesis and the canonical autophagic machinery. Whereas the fyve3 mutation alone barely affects autophagic flux, it suppresses defective autophagy in fyve2 mutants. Based on the molecular genetics and cell biological data, we propose that FYVE3 specifically regulates FYVE2-dependent autophagy

Delivery of Prolamins to the Protein Storage Vacuole in Maize Aleurone Cells[W]

This study uses a combination of molecular approaches, in vivo imaging of fluorescent proteins, and structural analysis by electron tomography to study the synthesis and transport of storage proteins in aleurone cells. It describes an unusual autophagic mechanism for the delivery of storage proteins to the vacuole that may be common to cereals

The Maize Zmsmu2 Gene Encodes a Putative RNA-Splicing Factor That Affects Protein Synthesis and RNA Processing during Endosperm Development1[W][OA]

We characterized two maize (Zea mays) mutants, zmsmu2-1 and zmsmu2-3, that result from insertion of a Mutator (Mu) transposable element in the first exon of a gene homologous to the nematode gene, smu-2, which is involved in RNA splicing. In addition to having a starchy endosperm with reduced levels of zein storage proteins, homozygous zmsmu2-1 mutants manifest a number of phenotypes, including defective meristem development. The zmsmu2 mutants have poor seedling viability and surviving plants are sterile. The gene encoding ZmSMU2 is expressed in the endosperm, embryo, and shoot apex, which explains the pleiotropic nature of the mutation. We found that proper expression of Zmsmu2 is required for efficient ribosomal RNA processing, ribosome biogenesis, and protein synthesis in developing endosperm. Based on the pleiotropic nature of the mutations and the known function of animal Zmsmu2 homologs, we propose a possible role for ZmSMU2 in the development of maize endosperm, as well as a mechanism by which misregulation of zmsmu2 causes the mutant phenotypes