P-type Pentatricopeptide Repeat Research Articles

A P-type pentatricopeptide repeat protein ZmRF5 promotes 5' region partial cleavages of atp6c transcripts to restore the fertility of CMS-C maize by recruiting a splicing factor.

A fast evolution within mitochondria genome(s) often generates discords between nuclear and mitochondria, which is manifested as cytoplasmic male sterility (CMS) and fertility restoration (Rf) system. The maize CMS-C trait is regulated by the chimeric mitochondrial gene, atp6c, and can be recovered by the restorer gene ZmRf5. Through positional cloning in this study, we identified the nuclear restorer gene, ZmRf5, which encodes a P-type pentatricopeptide repeat (PPR) family protein. The over-expression of ZmRf5 brought back the fertility to CMS-C plants, whereas its genomic editing by CRISPR/Cas9 induced abortive pollens in the restorer line. ZmRF5 is sorted to mitochondria, and recruited RS31A, a splicing factor, through MORF8 to form a cleaving/restoring complex, which promoted the cleaving of the CMS-associated transcripts atp6c by shifting the major cleavage site from 480th nt to 344 th nt for fast degradation, and preserved just right amount of atp6c RNA for protein translation, providing adequate ATP6C to assembly complex V, thus restoring male fertility. Interestingly, ATP6C in the sterile line CMo17A, with similar cytology and physiology changes to YU87-1A, was accumulated much less than it in NMo17B, exhibiting a contrary trend in the YU87-1 nuclear genome previously reported, and was restored to normal level in the presence of ZmRF5. Collectively these findings unveil a new molecular mechanism underlying fertility restoration by which ZmRF5 cooperates with MORF8 and RS31A to restore CMS-C fertility in maize, complemented and perfected the sterility mechanism, and enrich the perspectives on communications between nucleus and mitochondria.

A mitochondrion-associated PPR protein, WBG1, regulates grain chalkiness in rice.

Rice kernel quality has vital commercial value. Grain chalkiness deteriorates rice's appearance and palatability. However, the molecular mechanisms that govern grain chalkiness remain unclear and may be regulated by many factors. In this study, we identified a stable hereditary mutant, white belly grain 1 (wbg1), which has a white belly in its mature grains. The grain filling rate of wbg1 was lower than that of the wild type across the whole filling period, and the starch granules in the chalky part were oval or round and loosely arranged. Map-based cloning showed that wbg1 was an allelic mutant of FLO10, which encodes a mitochondrion-targeted P-type pentatricopeptide repeat protein. Amino acid sequence analysis found that two PPR motifs present in the C-terminal of WBG1 were lost in wbg1. This deletion reduced the splicing efficiency of nad1 intron 1 to approximately 50% in wbg1, thereby partially reducing the activity of complex I and affecting ATP production in wbg1 grains. Furthermore, haplotype analysis showed that WBG1 was associated with grain width between indica and japonica rice varieties. These results suggested that WBG1 influences rice grain chalkiness and grain width by regulating the splicing efficiency of nad1 intron 1. This deepens understanding of the molecular mechanisms governing rice grain quality and provides theoretical support for molecular breeding to improve rice quality.

Rice FLOURY ENDOSPERM22, encoding a pentatricopeptide repeat protein, is involved in both mitochondrial RNA splicing and editing and is crucial for endosperm development.

Most of the reported P-type pentatricopeptide repeat (PPR) proteins play roles in organelle RNA stabilization and splicing. However, P-type PPRs involved in both RNA splicing and editing have rarely been reported, and their underlying mechanism remains largely unknown. Here, we report a rice floury endosperm22 (flo22) mutant with delayed amyloplast development in endosperm cells. Map-based cloning and complementation tests demonstrated that FLO22 encodes a mitochondrion-localized P-type PPR protein. Mutation of FLO22 resulting in defective trans-splicing of mitochondrial nad1 intron 1 and perhaps causing instability of mature transcripts affected assembly and activity of complex Ⅰ, and mitochondrial morphology and function. RNA-seq analysis showed that expression levels of many genes involved in starch and sucrose metabolism were significantly down-regulated in the flo22 mutant compared with the wild type, whereas genes related to oxidative phosphorylation and the tricarboxylic acid cycle were significantly up-regulated. In addition to involvement in splicing as a P-type PPR protein, we found that FLO22 interacted with DYW3, a DYW-type PPR protein, and they may function synergistically in mitochondrial RNA editing. The present work indicated that FLO22 plays an important role in endosperm development and plant growth by participating in nad1 maturation and multi-site editing of mitochondrial messager RNA.

The small PPR protein SPR2 interacts with PPR-SMR1 to facilitate the splicing of introns in maize mitochondria.

Splicing of plant mitochondrial introns is facilitated by numerous nucleus-encoded protein factors. Although some splicing factors have been identified in plants, the mechanism underlying mitochondrial intron splicing remains largely unclear. In this study, we identified a small P-type pentatricopeptide repeat (PPR) protein containing merely four PPR repeats, small PPR protein 2 (SPR2), which is required for the splicing of more than half of the introns in maize (Zea mays) mitochondria. Null mutations of Spr2 severely impair the splicing of 15 out of the 22 mitochondrial Group II introns, resulting in substantially decreased mature transcripts, which abolished the assembly and activity of mitochondrial complex I. Consequently, embryogenesis and endosperm development were arrested in the spr2 mutants. Yeast two-hybrid, luciferase complementation imaging, bimolecular fluorescence complementation, and semi-in vivo pull-down analyses indicated that SPR2 interacts with small MutS-related domain protein PPR-SMR1, both of which are required for the splicing of 13 introns. In addition, SPR2 and/or PPR-SMR1 interact with other splicing factors, including PPR proteins EMPTY PERICARP16, PPR14, and chloroplast RNA splicing and ribosome maturation (CRM) protein Zm-mCSF1, which participate in the splicing of specific intron(s) of the 13 introns. These results prompt us to propose that SPR2/PPR-SMR1 serves as the core component of a splicing complex and possibly exerts the splicing function through a dynamic interaction with specific substrate recognizing PPR proteins in mitochondria.

The P-type pentatricopeptide repeat protein DWEORG1 is a non-previously reported rPPR protein of Arabidopsis mitochondria

Gene expression in plant mitochondria is mainly regulated by nuclear-encoded proteins on a post-transcriptional level. Pentatricopeptide repeat (PPR) proteins play a major role by participating in mRNA stability, splicing, RNA editing, and translation initiation. PPR proteins were also shown to be part of the mitochondrial ribosome (rPPR proteins), which may act as regulators of gene expression in plants. In this study, we focus on a mitochondrial-located P-type PPR protein—DWEORG1—from Arabidopsis thaliana. Its abundance in mitochondria is high, and it has a similar expression pattern as rPPR proteins. Mutant dweorg1 plants exhibit a slow-growth phenotype. Using ribosome profiling, a decrease in translation efficiency for cox2, rps4, rpl5, and ccmFN2 was observed in dweorg1 mutants, correlating with a reduced accumulation of the Cox2 protein in these plants. In addition, the mitochondrial rRNA levels are significantly reduced in dweorg1 compared with the wild type. DWEORG1 co-migrates with the ribosomal proteins Rps4 and Rpl16 in sucrose gradients, suggesting an association of DWEORG1 with the mitoribosome. Collectively, this data suggests that DWEORG1 encodes a novel rPPR protein that is needed for the translation of cox2, rps4, rpl5, and ccmFN2 and provides a stabilizing function for mitochondrial ribosomes.

Fine mapping and candidate gene analysis of qGSN5, a novel quantitative trait locus coordinating grain size and grain number in rice.

KEY MESSAGE: qGSN5, a novel quantitative trait locus coordinating grain size and grain number in rice, was fine-mapped to an 85.60-kb region. GS3 may be a suppressor of qGSN5. Grain size and grain number are two factors that directly determine rice grain yield; however, the underlying genetic mechanisms are complicated and remain largely unclear. In this study, a chromosome segment substitution line (CSSL), CSSL28, which showed increased grain size and decreased grain number per panicle, was identified in a set of CSSLs derived from a cross between 93-11 (recipient) and Nipponbare (donor). Four substitution segments were identified in CSSL28, and the substitution segment located on chromosome 5 was responsible for the phenotypes of CSSL28. Thus, we defined this quantitative trait locus (QTL) as grain size and grain number 5 (qGSN5). Cytological and quantitative PCR analysis showed that qGSN5 regulates the development of the spikelet hull by affecting cell proliferation. Genetic analysis showed that qGSN5 is a semi-dominant locus regulating grain size and grain number. Through map-based cloning and overlapping substitution segment analysis, qGSN5 was finally delimited to an 85.60-kb region. Based on sequence and quantitative PCR analysis, Os05g47510, which encodes a P-type pentatricopeptide repeat protein, is the most likely candidate gene for qGSN5. Pyramiding analysis showed that the effect of qGSN5 was significantly lower in the presence of a functional GS3 gene, indicating that GS3 may be a suppressor of qGSN5. In addition, we found that qGSN5 could improve the grain shape of hybrid rice. Together, our results lay the foundation for cloning a novel QTL coordinating grain size and grain number in rice and provide a good genetic material for long-grain hybrid rice breeding.

The main restorer Rf3 of maize S type cytoplasmic male sterility encodes a PPR protein that functions in reduction of the transcripts of orf355

Male sterile lines, especially cytoplasmic male sterile (CMS) lines, are extensively used in commercial hybrid seed production. Therefore a better understanding of the genetic basis of fertility restoration for CMS is important for the utility of heterosis in crops. In general, restorer of fertility (Rf) is controlled by one or two major genomic genes, and most of the Rf genes encode proteins containing P type pentatricopeptide repeat (PPR) motifs (Wang et al., 2006; Hu et al., 2012; Tang et al., 2014; Liu et al., 2016).

The pentatricopeptide repeat protein EMP603 is required for the splicing of mitochondrial Nad1 intron 2 and seed development in maize.

Intron splicing is an essential event in post-transcriptional RNA processing in plant mitochondria, which requires the participation of diverse nuclear-encoded splicing factors. However, it is presently unclear how these proteins cooperatively take part in the splicing of specific introns. In this study, we characterized a nuclear-encoded mitochondrial P-type pentatricopeptide repeat (PPR) protein named EMP603. This protein is essential for splicing of intron 2 in the Nad1 gene and interacts with the mitochondria-localized DEAD-box RNA helicase PMH2-5140, the RAD52-like proteins ODB1-0814 and ODB1-5061, and the CRM domain-containing protein Zm-mCSF1. Further study revealed that the N-terminal region of EMP603 interacts with the DEAD-box of PMH2-5140, the CRM domain of Zm-mCSF1, and OBD1-5061, but not with OBD1-0814, whereas the PPR domain of EMP603 can interact with ODB1-0814, ODB1-5061, and PMH2-5140, but not with Zm-mCSF1. Defects in EMP603 severely disrupt the assembly and activity of mitochondrial complex I, leading to impaired mitochondrial function, and delayed seed development. The interactions revealed between EMP603 and PMH2-5140, ODB1-0814, ODB1-5061, and Zm-mCSF1 indicate a possible involvement of a dynamic 'spliceosome-like' complex in intron splicing, and may accelerate the elucidation of the intron splicing mechanism in plant mitochondria.

In Arabidopsis thaliana mitochondria 5\u2032 end polymorphisms of nad4L-atp4 and nad3-rps12 transcripts are linked to RNA PROCESSING FACTORs 1 and 8

Key messageRNA PROCESSING FACTORs 1 AND 8 (RPF1 and RPF8), both restorer of fertility like pentatricopeptide repeat proteins, are required for processing of dicistronic nad4L-atp4 and nad3-rps12 transcripts in Arabidopsis mitochondria. In mitochondria of Arabidopsis thaliana (Arabidopsis), the 5′ termini of many RNAs are generated on the post-transcriptional level. This process is still poorly understood in terms of both the underlying mechanism as well as proteins required. Our studies now link the generation of polymorphic 5′ extremities of the dicistronic nad3-rps12 and nad4L-atp4 transcripts to the function of the P-type pentatricopeptide repeat proteins RNA PROCESSING FACTORs 8 (RPF8) and 1 (RPF1). RPF8 is required to generate the nad3-rps12 -141 5′ end in ecotype Van-0 whereas the RPF8 allele in Col has no function in the generation of any 5′ terminus of this transcript. This observation strongly suggests the involvement of an additional factor in the generation of the -229 5′ end of nad3-rps12 transcripts in Col. RPF1, previously found to be necessary for the generation of the -228 5′ end of the major 1538 nucleotide-long nad4 mRNAs, is also important for the formation of nad4L-atp4 transcripts with a 5′ end at position -318 in Col. Many Arabidopsis ecotypes contain inactive RPF1 alleles resulting in the accumulation of various low abundant nad4L-atp4 RNAs which might represent precursor and/or degradation products. Some of these ecotypes accumulate major, but slightly smaller RNA species. The introduction of RPF1 into these lines not only establishes the formation of the major nad4L-atp4 dicistronic mRNA with the -318 5′ terminus, the presence of this gene also suppresses the accumulation of most alternative nad4L-atp4 RNAs. Beside RPF1, several other factors contribute to nad4L-atp4 transcript formation.

A PPR Protein ACM1 Is Involved in Chloroplast Gene Expression and Early Plastid Development in Arabidopsis.

Chloroplasts cannot develop normally without the coordinated action of various proteins and signaling connections between the nucleus and the chloroplast genome. Many questions regarding these processes remain unanswered. Here, we report a novel P-type pentatricopeptide repeat (PPR) factor, named Albino Cotyledon Mutant1 (ACM1), which is encoded by a nuclear gene and involved in chloroplast development. Knock-down of ACM1 transgenic plants displayed albino cotyledons but normal true leaves, while knock-out of the ACM1 gene in seedlings was lethal. Fluorescent protein analysis showed that ACM1 was specifically localized within chloroplasts. PEP-dependent plastid transcript levels and splicing efficiency of several group II introns were seriously affected in cotyledons in the RNAi line. Furthermore, denaturing gel electrophoresis and Western blot experiments showed that the accumulation of chloroplast ribosomes was probably damaged. Collectively, our results indicate ACM1 is indispensable in early chloroplast development in Arabidopsis cotyledons.

Expanded Function of the P-Type Pentatricopeptide Repeat Protein ATP4 in RNA Editing.

Chloroplasts are semiautonomous organelles that retain their own genomes derived from their cyanobacterial ancestors, but with reduced size and gene number. However, chloroplast genes cannot function autonomously. Instead, nucleus-encoded factors are required for all steps of chloroplast gene

DEK43 is a P-type pentatricopeptide repeat (PPR) protein responsible for the Cis-splicing of nad4 in maize mitochondria.

Mitochondria, the main energy transducers in plant cells, require the proper assembly of respiratory chain complexes I-V for their function. The NADH dehydrogenase 4 (nad4) gene encodes mitochondrial respiratory chain complex I subunit IV, but the mechanism underlying nad4 transcript splicing is unclear. Here, we report that the P-type pentatricopeptide repeat (PPR) protein DEFECTIVE KERNEL 43 (DEK43) is responsible for cis-splicing of the nad4 transcript in maize. We demonstrate that DEK43 localizes to both the nucleus and mitochondria. The mutation of Dek43 resulted in embryo-lethal and light-colored defective kernels. Among the 22 mitochondrial group II introns, the splicing efficiency of nad4 introns 1 and 3 was reduced by up to 50% compared to the wild type. The levels of complex I and supercomplex I+III2 were also reduced in dek43. Furthermore, in-gel NADH dehydrogenase assays indicated that the activities of these complexes were significantly reduced in dek43. Further, the mitochondrial ultrastructure was altered in the mutant. Together, our findings indicate that DEK43, a dual-localized PPR protein, plays an important role in maintaining mitochondrial function and maize kernel development.

ZmSMK9, a pentatricopeptide repeat protein, is involved in the cis-splicing of nad5, kernel development and plant architecture in maize

Maize kernel size and weight are essential contributors to its yield. So the identification of the genes controlling kernel size and weight can give us a chance to gain the yield. Here, we identified a small kernel mutant, Zea mays small kernel 9 (Zmsmk9), in maize. Cytological observation showed that the development of the endosperm and embryo was delayed in Zmsmk9 mutants at the early stages, resulting in a small kernel phenotype. Interestingly, despite substantial variation in kernel size, the germination of Zmsmk9 seeds was comparable to that of WT, and could develop into normal plants with upright leaf architecture. We cloned Zmsmk9 via map-based cloning. ZmSMK9 encodes a P-type pentatricopeptide repeat protein that targets to mitochondria, and is involved in RNA splicing in mitochondrial NADH dehydrogenase5 (nad5) intron-1 and intron-4. Consistent with the delayed development phenotype, transcriptome analysis of 12-DAP endosperm showed that starch and zeins biosynthesis related genes were dramatically down regulated in Zmsmk9, while cell cycle and cell growth related genes were dramatically increased. As a result, ZmSMK9 is a novel gene required for the splicing of nad5 intron-1 and intron-4, kernel development, and plant architecture in maize.

EMB-7L is required for embryogenesis and plant development in maize involved in RNA splicing of multiple chloroplast genes

Embryo and endosperm originate from the double fertilization, but they have different developmental fates and biological functions. We identified a previously undescribed maize seed mutant, wherein the embryo appears to be more severely affected than the endosperm (embryo-specific, emb). In the W22 background, the emb embryo arrests at the transition stage whereas its endosperm appears nearly normal in size. At maturity, the embryo in W22-emb is apparently small or even invisible. In contrast, the emb endosperm develops into a relative normal size. We cloned the mutant gene on the Chromosome 7L and designated it emb-7L. This gene is generally expressed, but it has a relatively higher expression level in leaves. Emb-7L encodes a chloroplast-localized P-type pentatricopeptide repeat (PPR) protein, consistent with the severe chloroplast deficiency in emb-7L albino seedling leaves. Full transcriptome analysis of the leaves of WT and emb-7L seedlings reveals that transcription of chloroplast protein-encoding genes are dramatically variable with pre-mRNA intron splicing apparently affected in a tissue-dependent pattern and the chloroplast structure and activity were dramatically affected including chloroplast membrane and photosynthesis machinery component and synthesis of metabolic products (e.g., fatty acids, amino acids, starch).

Rice FLOURY ENDOSPERM10 encodes a pentatricopeptide repeat protein that is essential for the trans-splicing of mitochondrial nad1 intron 1 and endosperm development.

Endosperm, the major storage organ in cereal grains, determines grain yield and quality. Despite the fact that a role for P-type pentatricopeptide repeat (PPR) proteins in the regulation of endosperm development has emerged, molecular functions of many P-type PPR proteins remain obscure. Here, we report a rice endosperm defective mutant, floury endosperm10 (flo10), which developed smaller starch grains in starchy endosperm and abnormal cells in the aleurone layer. Map-based cloning and rescued experiments showed that FLO10 encodes a P-type PPR protein with 26 PPR motifs, which is localized to mitochondria. Loss of function of FLO10 affected the trans-splicing of the mitochondrial nad1 intron 1, which was accompanied by the increased accumulation of the nad1 exon 1 and exons 2-5 precursors. The failed formation of mature nad1 led to a dramatically decreased assembly and activity of complex I, reduced ATP production, and changed mitochondrial morphology. In addition, loss of function of FLO10 significantly induced an alternative respiratory pathway involving alternative oxidase. These results reveal that FLO10 plays an important role in the maintenance of mitochondrial function and endosperm development through its effect on the trans-splicing of the mitochondrial nad1 intron 1 in rice.

Two interacting PPR proteins are major Arabidopsis editing factors in plastid and mitochondria

RNA editing is converting hundreds of cytosines into uridines during organelle gene expression of land plants. The pentatricopeptide repeat (PPR) proteins are at the core of this posttranscriptional RNA modification. Even if a PPR protein defines the editing site, a DYW domain of the same or another PPR protein is believed to catalyze the deamination. To give insight into the organelle RNA editosome, we performed tandem affinity purification of the plastidial CHLOROPLAST BIOGENESIS 19 (CLB19) PPR editing factor. Two PPR proteins, dually targeted to mitochondria and chloroplasts, were identified as potential partners of CLB19. These two proteins, a P-type PPR and a member of a small PPR-DYW subfamily, were shown to interact in yeast. Insertional mutations resulted in embryo lethality that could be rescued by embryo-specific complementation. A transcriptome analysis of these complemented plants showed major editing defects in both organelles with a very high PPR type specificity, indicating that the two proteins are core members of E+-type PPR editosomes.

EMPTY PERICARP11 serves as a factor for splicing of mitochondrial nad1 intron and is required to ensure proper seed development in maize.

Group II introns are common in the mitochondrial genome of higher plant species. The splicing of these introns is a complex process involving the synergistic action of multiple factors. However, few of these factors have been characterized in maize. In this study, we found that the Empty pericarp11 (Emp11) gene, which encodes a P-type pentatricopeptide repeat (PPR) protein, is required for the development of maize seeds. The loss of Emp11 function seriously impairs embryo and endosperm development, resulting in empty pericarp seeds in maize, and alteration in Emp11 expression leads to quantitative variation in kernel size and weight. We found that the emp11 mutants showed a failure in nad1 intron splicing, exhibited a severe reduction in complex I assembly and activity, mitochondrial structure disturbances, and an increase in alternative oxidase AOX2 and AOX3 levels. Interestingly, the emp11 phenotype was very severe in the W22 inbred line but could be partially recovered in B73 BC2F2 segregating ears. These results suggest that EMP11 serves as a factor for the splicing of mitochondrial nad1 introns and is required for mitochondrial function to ensure proper seed development in maize.

Comparative genomic analysis of the compound Brassica napus Rf locus.

BackgroundThe plant trait of cytoplasmically-inherited male sterility (CMS) and its suppression by nuclear restorer-of-fertility (Rf) genes can be viewed as a genetic arms race between the mitochondrial and nuclear genomes. Most nuclear Rf genes have been shown to encode P-type pentatricopeptide repeat proteins (PPRs). Phylogenetic analysis of P-class PPRs from sequenced plants genomes has shown that Rf-proteins cluster in a distinct clade of P-class PPRs, RFL-PPRs, that display hallmarks of positive evolutionary selection. Genes encoding RFL-PPRs (RFLs) within a given plant genome tend to be closely related both in sequence and position, but a detailed understanding of how such species-specific expansion occurs is lacking. In the canola, (oilseed rape) species Brassica napus, previous work has indicated the nuclear restorer genes for the two native forms of CMS, Rfn (for nap CMS) and Rfp (pol CMS), represent alternate haplotypes, or alleles, of a single nuclear locus.ResultsFine genetic mapping indicates that Rfn does indeed localize to the same genomic region as Rfp. We find this region is enriched in RFL genes, three of which, based on their position and expression, represent potential candidates for Rfn; one of these genes, designated PPR4, is a preferred candidate in that it is not expressed in the nap CMS line. Comparison of the corresponding regions of the genomes of B. rapa, B. oleracea, Arabidopsis thaliana and A. lyrata provides insight into the expansion of this group of RFL genes in different lines of evolutionary descent.ConclusionsUnlike other nuclear restorer loci containing multiple RFL genes, the RFL genes in the Rf region of B. napus are not present in tandem arrays but rather are dispersed in genomic location. The genes do not share similar flanking non-coding regions and do not contain introns, indicating that they have duplicated primarily through a retrotransposition-mediated process. In contrast, segmental duplication has been responsible for the distribution of the 10 sequences we annotated as RFL genes in the corresponding region of the A. lyrata genome. Our observations define the Brassica Rf locus and indicate that different mechanisms may be responsible for the proliferation of RFL genes even among closely related genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3117-0) contains supplementary material, which is available to authorized users.

EMPTY PERICARP16 is required for mitochondrial nad2 intron 4 cis-splicing, complex I assembly and seed development in maize.

In higher plants, chloroplast and mitochondrial transcripts contain a number of group II introns that need to be precisely spliced before translation into functional proteins. However, the mechanism of splicing and the factors involved in this process are not well understood. By analysing a seed mutant in maize, we report here the identification of Empty pericarp16 (Emp16) that is required for splicing of nad2 intron 4 in mitochondria. Disruption of Emp16 function causes developmental arrest in the embryo and endosperm, giving rise to an empty pericarp phenotype in maize. Differentiation of the basal endosperm transfer layer cells is severely affected. Molecular cloning indicates that Emp16 encodes a P-type pentatricopeptide repeat (PPR) protein with 11 PPR motifs and is localized in the mitochondrion. Transcript analysis revealed that mitochondrial nad2 intron 4 splicing is abolished in the emp16 mutants, leading to severely reduced assembly and activity of complex I. In response, the mutant dramatically increases the accumulation of mitochondrial complex III and the expression of alternative oxidase AOX2. These results imply that EMP16 is specifically required for mitochondrial nad2 intron 4 cis-splicing and is essential for complex I assembly and embryogenesis and development endosperm in maize.