Wilhelm Gruissem

Fruits: A Developmental Perspective.

Embryonic development in many angiosperms occurs concomitantly with the development of the ovary into a specialized organ, the fruit, which provides a suitable environment for seed maturation and often a mechanism for the dispersa1 of mature seeds, as Darwin observed. Despite centuries of intensive genetic selection of agriculturally valuable fruit, we still lack most information about how fruits develop, how this development is coordinated with embryonic development and seed formation, and the molecular, cellular, and physiological events that control fruit growth and differentiation. The last 10 years have seen a rapid surge of information on one commercially important aspect of fruit development, fruit ripening, including the genetic control of temporal events during the ripening phase (Theologis, 1992; Theologis et al., 1992). However, fewer advances have been made on temporal and spatial controls of fruit set and growth, although from the agricultura1 point of view, these aspects are of equally critical importance. We will provide a perspective on the molecular, cellular, and physiological mechanisms that must be considered as integral parts of the fruit developmental process. The discussion below will illustrate that fruit development is a potentially useful system to learn more about complex regulatory mechanisms that control the division, growth, and differentiation of plant cells.

Control of plastid gene expression: 3′ inverted repeats act as mRNA processing and stabilizing elements, but do not terminate transcription

We have examined the function of inverted repeat sequences found at the 3 ends of plastid DNA transcription units in higher plants, using a homologous in vitro transcription extract. The inverted repeat sequences are ineffective as transcription terminators, but serve as efficient RNA processing elements. Synthetic RNAs are processed in a 3-5 direction by a nuclease activity present in the transcription extract, generating nearly homogeneous 3 ends distal to the inverted repeat sequence. S1 nuclease protection experiments demonstrate that the 3 ends generated in vitro coincide with those found for plastid mRNAs in vivo. RNA molecules possessing inverted repeats near their 3 ends are substantially more stable than control RNAs in the chloroplast extract, and kinetic measurements indicate that each RNA has a unique decay rate. Coupled with previously published information suggesting that the differential accumulation of plastid RNAs during development is effectively controlled by post-transcriptional mechanisms, these results raise the possibility that RNA processing and stability, specifically involving 3 end inverted repeats, are important regulatory features of plastid gene expression.

Control of plastid gene expression during development: The limited role of transcriptional regulation

We have analyzed the transcriptional regulation of plastid genes during chloroplast development in illuminated spinach cotyledons and during leaf formation. The RNAs encoded by plastid genes accumulate with different kinetics during the developmental transitions. Using a novel plastid run-on transcription assay we demonstrate that the transcriptional regulation of a large, diverse group of chloroplast genes is of relatively minor importance for the control of their expression. The general transcriptional activity of the plastid genome increases after illumination and decreases during leaf development. This modulation of general transcriptional activity affects most plastid genes simultaneously and is not correlated with adjustments of the plastid DNA copy number. There are no major changes in the relative transcriptional activities of different genes, although their steady-state mRNA levels change dramatically. The analysis of ten specific plastid genes shows that their relative transcriptional activities are largely maintained throughout the developmental program. This limited transcriptional regulation suggests that plastid gene expression in higher plants is effectively controlled at the posttranscriptional level.

RRB1 AND RRB2 ENCODE MAIZE RETINOBLASTOMA-RELATED PROTEINS THAT INTERACT WITH A PLANT D-TYPE CYCLIN AND GEMINIVIRUS REPLICATION PROTEIN

Unlike mammalian and yeast cells, little is known about how plants regulate G1 progression and entry into the S phase of the cell cycle. In mammalian cells, a key regulator of this process is the retinoblastoma tumor suppressor protein (RB). In contrast, G1 control in Saccharomyces cerevisiae does not utilize an RB-like protein. We report here the cloning of cDNAs from two Zea mays genes, RRB1 and RRB2, that encode RB-related proteins. Further, RRB2 transcripts are alternatively spliced to yield two proteins with different C termini. At least one RRB gene is expressed in all the tissues examined, with the highest levels seen in the shoot apex. RRB1 is a 96-kDa nuclear protein that can physically interact with two mammalian DNA tumor virus oncoproteins, simian virus 40 large-T antigen and adenovirus E1A, and with a plant D-type cyclin. These associations are abolished by mutation of a conserved cysteine residue in RRB1 that is also essential for RB function. RRB1 binding potential is also sensitive to deletions in the conserved A and B domains, although differences exist in these effects compared to those of human RB. RRB1 can also bind to the AL1 protein from tomato golden mosaic virus (TGMV), a protein which is essential for TGMV DNA replication. These results suggest that G1 regulation in plant cells is controlled by a mechanism which is much more similar to that found in mammalian cells than that in yeast.

Chloroplast gene expression: How plants turn their plastids on

Wilhelm Gruissem Department of Botany University of California Berkeley, California 94720 Plant and animal cells are fundamentally very similar. Be- sides the organelles found in both cell types, however, plant cells contain a unique class of organelles, the plastids. Since the early discovery by Correns (1909) and Baur (1909) that mutations affecting plastid phenotypes in higher plants frequently exhibit non-Mendelian inheri- tance, research on the DNA of this organelle has now yielded the complete sequence of the plastid genomes from tobacco (Shinozaki et al., 1986) and liverwort (Oh- yama et al., 1986). Plastids exist in a number of different forms with different functions, but the green chloroplast was the first to be discovered, and is the best studied of all plastids. The diversity of plastid types is controlled by the developmental program of the plant, which indicates that there must be a significant flow of information be- tween two separate genetic compartments in the cell. The use of chloroplasts to study photosynthesis and the in- tricacy of photosynthetic complexes has yielded new infor- mation on controls of organelle gene expression and the communication of different genomes in eukaryotic cells. In developing plants, chloroplasts are derived from small proplastids, which are the undifferentiated plastids present in meristematic cells. During the development of chloroplasts in photosynthetic tissues, photosynthetic electron-transfer components are assembled into pho- tosystems I and II, cytochrome bsf, and ATP synthase complexes, each of which consists of up to 20 polypep- tides. Proplastids and chloroplasts can also differentiate into specialized plastid types that assume other functions in nonphotosynthetic plant organs of higher plants, such as amyloplasts in roots and tubers or chromoplasts in many flowers and fruits. Photosynthesis, together with other plastid functions, requires the products of several hundred genes, of which only about 120 are present in the approximately 150 kb chloroplast genome. All other plas- tid proteins are expressed from nuclear genes. The devel- opment and differentiation of photosynthetically compe- tent chloroplasts and other plastid types thus present a challenging opportunity: to decipher how plastid gene ex- pression is controlled temporally and spatially in different plant organs, and also in coordination with the expression of nuclear genes for chloroplast proteins. Initial efforts to analyze the controls of plastid gene expression have con- centrated on the transcription of genes for photosynthetic proteins and tRNAs. Recent progress appears to support a model that places a major emphasis on posttranscrip- tional and translational regulatory mechanisms. In con- trast, known nuclear genes for photosynthetic proteins ap- pear to be regulated primarily at the level of transcription. The purpose of this review is to discuss some of the cen- tral problems and ideas in the field of chloroplast gene ex- pression, not to provide a comprehensive review on all that is known. (For further information on chloroplast ge- nome structures, genes, and transcriptional and transla- tional components, readers should consult Whitfeld and Bottomley, 1983; Ellis, 1984; Sugiura, 1987; Umesono and Ozeki, 1987; Gruissem, 1989; Mullet, 1988; Bonham- Smith and Bourque, 1988.) Linkage of Genes in Many Chloroplast Transcription Units Is Conserved Compared with the small number of genes in animal, fun- gal and plant mitochondria, the chloroplast genome con- tains a substantially larger number of genes, encoding both genetic and photosynthetic functions. The genes identified thus far include a complete set of 30 tRNAs, four ribosomal RNAs (23S, 16S, 5S, and 4.5s) and 20 ribo- somal proteins. Twenty-two genes encode proteins for thylakoid membrane complexes (photosystem I, photosys- tern II, cytochrome bsf complex, and ATP synthase), and the sequences of six other open reading frames share similarities with the mitochondrial genes for the subunits of the human respiratory chain NADH dehydrogenase. Several of the remaining unidentified reading frames are conserved between diverse species, which suggests that they may also encode functional plastid polypeptides. Most plastid genes are organized into polycistronic tran- scription units reminiscent of bacterial operons. The se- quence analysis of the entire tobacco and liverwort chlo- roplast genomes (Shinozaki et al., 1986; Ohyama et al., 1986) together with the partial sequence and mapping data from other plant chloroplast genomes, has revealed that the arrangement of genes within these transcription units is highly conserved, although transcription are extensively rearranged in some plant species (reviewed by Palmer, 1985). Detailed mapping of chloroplast DNAs from pea and geranium, for example, has found that such rearrangements involve primarily inversions of large clus- ters of genes. Most, but not all, the genes linked in these clusters are cotranscribed. It has been possible, at least some cases, to trace the linkage of chloroplast gene sets to the cyanobacterial genome (Cozens et al., 1986) which is the putative ancestral genome of chloroplast genome. However, the conserved arrangement of genes in plant chloroplast genomes is not found algae, for which Chlamydomonas and Euglena are the best studied examples, possibly indicating different endosymbiotic events. Chloroplast RNA Polymerases and Promoter Regions The possibility that transcriptional regulation chlo- roplast genes could be a key control during chloroplast development in plants spurred early investigations into the transcriptional components of this organelle. Applica- tion of different schemes for preparing DNA-dependent RNA polymerase from chloroplasts led to the intriguing idea that chloroplasts of algae and plants may contain at least two different RNA polymerase activities distinguish-

A geminivirus replication protein interacts with the retinoblastoma protein through a novel domain to determine symptoms and tissue specificity of infection in plants.

Geminiviruses replicate in nuclei of mature plant cells after inducing the accumulation of host DNA replication machinery. Earlier studies showed that the viral replication factor, AL1, is sufficient for host induction and interacts with the cell cycle regulator, retinoblastoma (pRb). Unlike other DNA virus proteins, AL1 does not contain the pRb binding consensus, LXCXE, and interacts with plant pRb homo logues (pRBR) through a novel amino acid sequence. We mapped the pRBR binding domain of AL1 between amino acids 101 and 180 and identified two mutants that are differentially impacted for AL1–pRBR interactions. Plants infected with the E‐N140 mutant, which is wild‐type for pRBR binding, developed wild‐type symptoms and accumulated viral DNA and AL1 protein in epidermal, mesophyll and vascular cells of mature leaves. Plants inoculated with the KEE146 mutant, which retains 16% pRBR binding activity, only developed chlorosis along the veins, and viral DNA, AL1 protein and the host DNA synthesis factor, proliferating cell nuclear antigen, were localized to vascular tissue. These results established the importance of AL1–pRBR interactions during geminivirus infection of plants.

Control mechanisms of plastid gene expression

Abstract Plastid DNAs of higher plants contain approximately 150 genes that encode RNAs and proteins for genetic and photosynthetic functions of the organelle. Results published in the last few years illustrate that the spatial and temporal expression of these plastid genes is regulated, in part, at the transcriptional level, but that developmentally controlled changes in mRNA stability, translational activity, and protein phosphorylation also have an important role in the control of plastid functions. This comprehensive review summarizes and discusses the mechanisms by which regulation of gene expression is exerted at the transcriptional and post‐transcriptional levels. It provides an overview of our current knowledge, but also emphasizes areas that are controversial and in which information on regulatory mechanisms is still incomplete.

The prenylation status of a novel plant calmodulin directs plasma membrane or nuclear localization of the protein.

Post‐translational attachment of isoprenyl groups to conserved cysteine residues at the C‐terminus of a number of regulatory proteins is important for their function and subcellular localization. We have identified a novel calmodulin, CaM53, with an extended C‐terminal basic domain and a CTIL CaaX‐box motif which are required for efficient prenylation of the protein in vitro and in vivo. Ectopic expression of wild‐type CaM53 or a non‐prenylated mutant protein in plants causes distinct morphological changes. Prenylated CaM53 associates with the plasma membrane, but the non‐prenylated mutant protein localizes to the nucleus, indicating a dual role for the C‐terminal domain. The subcellular localization of CaM53 can be altered by a block in isoprenoid biosynthesis or sugar depletion, suggesting that CaM53 activates different targets in response to metabolic changes. Thus, prenylation of CaM53 appears to be a novel mechanism by which plant cells can coordinate Ca2+ signaling with changes in metabolic activities.

Changes in Chloroplast mRNA Stability during Leaf Development.

During spinach leaf development, chloroplast-encoded mRNAs accumulate to different steady-state levels. Their relative transcription rates alone, however, cannot account for the changes in mRNA amount. In this study, we examined the importance of mRNA stability for the regulation of plastid mRNA accumulation using an in vivo system to measure mRNA decay in intact leaves by inhibiting transcription with actinomycin D. Decay of psbA and rbcL mRNAs was assayed in young and mature leaves. The psbA mRNA half-life was increased more than twofold in mature leaves compared with young leaves, whereas rbcL mRNA decayed with a similar relative half-life at both leaf developmental stages. The direct in vivo measurements demonstrated that differential mRNA stability in higher plant plastids can account for differences in mRNA accumulation during leaf development. The role of polysome association in mRNA decay was also investigated. Using organelle-specific translation inhibitors that force mRNAs into a polysome-bound state or deplete mRNAs of ribosomes, we measured mRNA decay in vivo in either state. The results showed that rbcL and psbA mRNAs are less stable when bound to polysomes relative to the polysome-depleted mRNAs and that their stabilities are differentially affected by binding to polysomes. The results suggested that ribosome binding and/or translation of the psbA and rbcL mRNAs may function to modulate the rate of their decay in chloroplasts.

Genomic organization, sequence analysis and expression of all five genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato

SummaryWe have cloned and sequenced all five members of the gene family for the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato, Lycopersicon esculentum cv. VFNT LA 1221 cherry line. Two of the five genes, designated Rbcs-1 and Rbcs-2, are present as single genes at individual loci. Three genes, designated Rbcs-3A, Rbcs-3B and Rbcs-3C, are organized in a tandem array within 10 kb at a third independent locus. The Rbcs-2 gene contains three introns; all the other members of the tomato gene family contain two introns. The coding sequence of Rbcs-1 differs by 14.0% from that of Rbcs-2 and by 13.3% from that of Rbcs-3 genes. Rbcs-2 shows 10.4% divergence from Rbcs-3. The exon and intron sequences of Rbcs-3A are identical to those of Rbcs-3C, and differ by 1.9% from those of Rbcs-3B. Nucleotide sequence analysis suggests that the five rbcS genes encode four different precursors, and three different mature polypeptides. S1 nuclease mapping of the 5′ end of rbcS mRNAs revealed that the mRNA leader sequences vary in length from 8 to 75 nucleotides. Northern analysis using gene-specific oligonucleotide probes from the 3′ non-coding region of each gene reveals a four to five-fold difference among the five genes in maximal steady-state mRNA levels in leaves.