Maureen R. Hanson

Interactions of Mitochondrial and Nuclear Genes That Affect Male Gametophyte Development

Apart from their agronomic importance in hybrid seed production, mutations that encode cytoplasmic male sterility (CMS) provide a means to probe the role of the mitochondrion in reproductive development. Fertility restorers are examples of nuclear genes that affect cytoplasmic gene expression, and

A pentatricopeptide repeat-containing gene restores fertility to cytoplasmic male-sterile plants

Known in over 150 species, cytoplasmic male sterility is encoded by aberrant mitochondrial genes that prevent pollen development. The RNA- or protein-level expression of most of the mitochondrial genes encoding cytoplasmic male sterility is altered in the presence of one or more nuclear genes called restorers of fertility that suppress the male-sterile phenotype. Cytoplasmic male sterility/restorer systems have been proven to be an invaluable tool in the production of hybrid seeds. Despite their importance for both the production of major crops such as rice and sunflower and the study of organelle/nuclear interactions in plants, none of the nuclear fertility-restorer genes that reduce the expression of aberrant mitochondrial proteins have previously been cloned. Here we report the isolation of a gene directly involved in the control of the expression of a cytoplasmic male sterility-encoding gene. The Petunia restorer of fertility gene product is a mitochondrially targeted protein that is almost entirely composed of 14 repeats of the 35-aa pentatricopeptide repeat motif. In a nonrestoring genotype we identified a homologous gene that exhibits a deletion in the promoter region and is expressed in roots but not in floral buds.

Mobilization of Rubisco and Stroma-Localized Fluorescent Proteins of Chloroplasts to the Vacuole by an ATG Gene-Dependent Autophagic Process

During senescence and at times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, Rubisco. While bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have visualized the fate of Rubisco, stroma-targeted green fluorescent protein (GFP) and DsRed, and GFP-labeled Rubisco in order to investigate the involvement of autophagy in the mobilization of stromal proteins to the vacuole. Using immunoelectron microscopy, we previously demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves. When leaves of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing stroma-targeted fluorescent proteins were incubated with concanamycin A to inhibit vacuolar H+-ATPase activity, spherical bodies exhibiting GFP or DsRed fluorescence without chlorophyll fluorescence were observed in the vacuolar lumen. Double-labeled immunoelectron microscopy with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. RCBs could also be visualized using GFP-labeled Rubisco directly. RCBs were not observed in leaves of a T-DNA insertion mutant in ATG5, one of the essential genes for autophagy. Stroma-targeted DsRed and GFP-ATG8 fusion proteins were observed together in autophagic bodies in the vacuole. We conclude that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction.

Redesigning photosynthesis to sustainably meet global food and bioenergy demand

The world’s crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.

Functioning and Variation of Cytoplasmic Genomes: Lessons from Cytoplasmic–Nuclear Interactions Affecting Male Fertility in Plants

Publisher Summary The generality of cytoplasmic male sterility (CMS) suggests that understanding its basis may give the fundamental information about cytoplasmic genomes functioning in plant development. This chapter describes the male sterility in Petunia, maize, sorghum, sugarbeet, Nicotiana, Vicia faba, sunflower, Solanum, cruciferous species, pearl millet, carrot, wheat and rye, and barley. In some CMS systems, identification of a CMS-specific DNA sequence and its products may immediately reveal why microspores or male reproductive tissues do not develop properly. However, mere identification of a CMS-correlated gene and protein of unknown function would not reveal the mechanism of CMS without further cytological, biochemical, and physiological studies. As functions of mitochondrial-encoded polypeptides have become known, new models for CMS mechanisms may arise. Such models could most readily be tested if cloned Mitochondrial DNA (mtDNA) sequences could be introduced anew into mitochondrial genomes. The underlying mechanisms of CMS are likely to be as diverse as the abnormal reproductive phenotypes observed within and among species.

A faster Rubisco with potential to increase photosynthesis in crops

In photosynthetic organisms, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating atmospheric CO2 into the biosphere. Owing to the wasteful oxygenase activity and slow turnover of Rubisco, the enzyme is among the most important targets for improving the photosynthetic efficiency of vascular plants. It has been anticipated that introducing the CO2-concentrating mechanism (CCM) from cyanobacteria into plants could enhance crop yield. However, the complex nature of Rubisco’s assembly has made manipulation of the enzyme extremely challenging, and attempts to replace it in plants with the enzymes from cyanobacteria and red algae have not been successful. Here we report two transplastomic tobacco lines with functional Rubisco from the cyanobacterium Synechococcus elongatus PCC7942 (Se7942). We knocked out the native tobacco gene encoding the large subunit of Rubisco by inserting the large and small subunit genes of the Se7942 enzyme, in combination with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal protein, CcmM35, which incorporates three small subunit-like domains. Se7942 Rubisco and CcmM35 formed macromolecular complexes within the chloroplast stroma, mirroring an early step in the biogenesis of cyanobacterial β-carboxysomes. Both transformed lines were photosynthetically competent, supporting autotrophic growth, and their respective forms of Rubisco had higher rates of CO2 fixation per unit of enzyme than the tobacco control. These transplastomic tobacco lines represent an important step towards improved photosynthesis in plants and will be valuable hosts for future addition of the remaining components of the cyanobacterial CCM, such as inorganic carbon transporters and the β-carboxysome shell proteins.

Novel composition of mitochondrial genomes in Petunia somatic hybrids derived from cytoplasmic male sterile and fertile plants

SummaryThe mitochondrial genomes of petunia somatic hybrid plants, which were derived from the fusion of male fertile P. hybrida protoplasts with cytoplasmic male sterile P. parodii protoplasts, were analyzed by endonuclease restriction and Southern blot hybridization analyses. We studied sterile and fertile somatic hybrids to address two main questions. First, is there any correlation between the mitochondrial DNA restriction banding patterns of the sterile and fertile parents and the banding patterns of the respective sterile and fertile somatic hybrids? Second, does the structure of somatic hybrid mitochondrial genomes differ from the parental mitochondrial genomes?We identified no clear sterile-specific correlation between the mitochondrial DNA restriction patterns of cytoplasmic male sterile somatic hybrids and those of the male sterile parent. Similarly, we found no clear relationship between the mitochondrial DNA restriction patterns of male fertile somatic hybrids and those of the male fertile parent. In view of this finding and the evidence that cytoplasmic male sterility and fertility segregate in somatic hybrids of petunia (Izhar et al. 1983) the nature of cytoplasmic male sterility in petunia differs significantly from cytoplasmic male sterility in tobacco (Gerstel 1980).Restriction fragment patterns show that the somatic hybrid mitochondrial genomes differ from each other and from both parents. Somatic hybrid mitochondrial genomes consist of DNA fragments derived from both parents in novel combinations. Hybridization data revealed the fates of parental restriction fragments in the somatic hybrids. Parental fragments may be present or absent in all somatic hybrids analyzed, or they may be present in some somatic hybrids and absent in others. These data are consistent with two non-exclusive possibilities. Separate DNA molecules might assort following protoplast fusion or intermolecular recombination might occur following protoplast fusion.

RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing

Transcripts of plant organelle genes are modified by cytidine-to-uridine (C-to-U) RNA editing, often changing the encoded amino acid predicted from the DNA sequence. Members of the PLS subclass of the pentatricopeptide repeat (PPR) motif-containing family are site-specific recognition factors for either chloroplast or mitochondrial C targets of editing. However, other than PPR proteins and the cis-elements on the organelle transcripts, no other components of the editing machinery in either organelle have previously been identified. The Arabidopsis chloroplast PPR protein Required for AccD RNA Editing 1 (RARE1) specifies editing of a C in the accD transcript. RARE1 was detected in a complex of >200 kDa. We immunoprecipitated epitope-tagged RARE1, and tandem MS/MS analysis identified a protein of unknown function lacking PPR motifs; we named it RNA-editing factor interacting protein 1 (RIP1). Yeast two-hybrid analysis confirmed RIP1 interaction with RARE1, and RIP1-GFP fusions were found in both chloroplasts and mitochondria. Editing assays for all 34 known Arabidopsis chloroplast targets in a rip1 mutant revealed altered efficiency of 14 editing events. In mitochondria, 266 editing events were found to have reduced efficiency, with major loss of editing at 108 C targets. Virus-induced gene silencing of RIP1 confirmed the altered editing efficiency. Transient introduction of a WT RIP1 allele into rip1 improved the defective RNA editing. The presence of RIP1 in a protein complex along with chloroplast editing factor RARE1 indicates that RIP1 is an important component of the RNA editing apparatus that acts on many chloroplast and mitochondrial C targets.

Identification of a mitochondrial protein associated with cytoplasmic male sterility in petunia.

The petunia fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS. To determine whether the pcf gene is expressed at the protein level, we produced antibodies to synthetic peptides specified by the coxII and urfS portions of the pcf gene. Anti-COXII peptide antibodies recognized petunia COXII but no other mitochondrial proteins. Anti-URF-S peptide antibodies recognized a 20-kilodalton protein present in both cytoplasmic male sterile and fertile lines and a protein with an apparent molecular mass of 25 kilodaltons present only in cytoplasmic male sterile lines. The 25-kilodalton protein was found to be synthesized by isolated mitochondria and to fractionate into both the soluble and membrane portions of disrupted mitochondria, whereas the 20-kilodalton protein was found only in the membrane fraction. The abundance of the 25-kilodalton protein was much lower in fertile plants carrying the cytoplasmic male sterile cytoplasm and a single dominant nuclear fertility restorer gene, Rf. Thus, the pcf gene is correlated with cytoplasmic male sterility not only by its co-segregation with the phenotype in somatic hybrids, but also by the modification of its expression at the protein level through the action of a nuclear gene that confers fertility.

Stromules and the dynamic nature of plastid morphology

Investigation of plastids via green fluorescent protein (GFP) has led to the rediscovery of tubular extensions of the plastid membrane, termed stromules, for stroma‐filled tubules. These unique structures are challenging our understanding of plastid structure and function. Stromules are highly dynamic, branching and elongating across the plant cell. Recent experiments indicate that cytoplasmic microtubules and microfilaments control the shape and motility of stromules. Whether stromule formation involves plastid‐specific structural systems, such as the plastid division machinery, remains open to debate. Fluorescence photobleaching experiments have revealed that GFP can traffic between plastids joined by stromules. As a result, interest has grown in whether other macromolecules can also travel through these connections. Although the function of stromules is unknown, several aspects of their biology suggest they play a role in molecular exchange between plastids and other organelles.