Peter Westhoff

The Sorghum bicolor genome and the diversification of grasses

Sorghum, an African grass related to sugar cane and maize, is grown for food, feed, fibre and fuel. We present an initial analysis of the ∼730-megabase Sorghum bicolor (L.) Moench genome, placing ∼98% of genes in their chromosomal context using whole-genome shotgun sequence validated by genetic, physical and syntenic information. Genetic recombination is largely confined to about one-third of the sorghum genome with gene order and density similar to those of rice. Retrotransposon accumulation in recombinationally recalcitrant heterochromatin explains the ∼75% larger genome size of sorghum compared with rice. Although gene and repetitive DNA distributions have been preserved since palaeopolyploidization ∼70 million years ago, most duplicated gene sets lost one member before the sorghum–rice divergence. Concerted evolution makes one duplicated chromosomal segment appear to be only a few million years old. About 24% of genes are grass-specific and 7% are sorghum-specific. Recent gene and microRNA duplications may contribute to sorghum’s drought tolerance.

VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation

The conversion of light to chemical energy by the process of photosynthesis is localized to the thylakoid membrane network in plant chloroplasts. Although several pathways have been described that target proteins into and across the thylakoids, little is known about the origin of this membrane system or how the lipid backbone of the thylakoids is transported and fused with the target membrane. Thylakoid biogenesis and maintenance seem to involve the flow of membrane elements via vesicular transport. Here we show by mutational analysis that deletion of a single gene called VIPP1 (vesicle-inducing protein in plastids 1) is deleterious to thylakoid membrane formation. Although VIPP1 is a hydrophilic protein it is found in both the inner envelope and the thylakoid membranes. In VIPP1 deletion mutants vesicle formation is abolished. We propose that VIPP1 is essential for the maintenance of thylakoids by a transport pathway not previously recognized.

Nucleotide sequence of the clustered genes for the 44 kd chlorophyll a apoprotein and the “32 kd”-like protein of the photosystem II reaction center in the spinach plastid chromosome

SummaryA 2,900 base pair DNA segment of the spinach plastid chromosome which encodes the genes for the 44 kd chlorophyll a apoprotein and a “32 kd”-like protein of the photosystem II reaction center has been subjected to sequence and Northern blot analysis. The genes are located almost centrally in the large single-copy segment of the chromosome adjacent to the two genes for the P700 chlorophyll a apoproteins of the photosystem I reaction center. The DNA sequence reveals two uninterrupted protein-coding regions of 473 (44 kd chlorophyll a apoprotein) and 353 triplets (“32 kd”-like protein). The latter gene is strikingly similar to the gene for the herbicide-binding “32 kd” protein which maps some 30 kbp distant on the plastid chromosome. The two genes overlap by 50 base pairs but are read in different phases. They may be contranscribed and the RNA modified to give several discrete species ranging in size from 1.6 to 4.6 kb. A presumptive promoter site was only identified for the “32 kd”-like protein, while potential ribosome binding and transcription termination sites are found preceding and following both genes, respectively. The polypeptides possess a high content of hydrophobic amino acids, most of which appear to be clustered in transmembrane spans. The molecular weights of 51,785 (44 kd chlorophyll a apoprotein) and 39,465 (“32 kd”-like protein) derived from the deduced amino acid sequences are higher than the experimentally determined protein sizes. Amino acid codon usage for both genes is highly selective. Comparison of the chlorophyll a apoproteins of spinach reveals regions of sequence homology.

A nuclear‐encoded protein of prokaryotic origin is essential for the stability of photosystem II in Arabidopsis thaliana

To understand the regulatory mechanisms underlying the biogenesis of photosystem II (PSII) we have characterized the nuclear mutant hcf136 of Arabidopsis thaliana and isolated the affected gene. The mutant is devoid of any photosystem II activity, and none of the nuclear‐ and plastome‐encoded subunits of this photosystem accumulate to significant levels. Protein labelling studies in the presence of cycloheximide showed that the plastome‐encoded PSII subunits are synthesized but are not stable. The HCF136 gene was isolated by virtue of its T‐DNA tag, and its identity was confirmed by complementation of homozygous hcf136 seedlings. Immunoblot analysis of fractionated chloroplasts showed that the HCF136 protein is a lumenal protein, found only in stromal thylakoid lamellae. The HCF136 protein is produced already in dark‐grown seedlings and its levels do not increase dramatically during light‐induced greening. This accumulation profile confirms the mutational data by showing that the HCF136 protein must be present when PSII complexes are made. HCF136 homologues are found in the cyanobacterium Synechocystis species PCC6803 (slr2034) and the cyanelle genome of Cyanophora paradoxa (ORF333), but are lacking in the plastomes of chlorophytes and metaphytes as well as from those of rhodo‐ and chromophytes. We conclude that HCF136 encodes a stability and/or assembly factor of PSII which dates back to the cyanobacterial‐like endosymbiont that led to the plastids of the present photosynthetic eukaryotes.

Isolation of high-chlorophyll-fluorescence mutants ofArabidopsis thaliana and their characterisation by spectroscopy, immunoblotting and Northern hybridisation

Thirty-four recessive photosynthetic mutants of the high-chlorophyll-fluorescence (hcf) phenotype have been isolated by screening 7700 M2 progenies of ethyl methane sulfonate-treated seeds ofArabidopsis thaliana. Most of the mutants isolated were found to be seedlinglethal, but could be grown on sucrose-supplemented media. Chlorophyll (Chl) fluorescence induction, absorption changes in the reaction-centre chlorophyll of PS I (P700) at 830 nm and Chla/Chlb ratios were recorded in order to probe the photosynthetic functions and to define the mutational lesion. These studies were complemented by immunoblot and Northern analyses which finally led to the classification of the mutants into six different groups. Four classes of mutants were affected in PS I, PS II (two different classes) or the intersystem electron-transport chain, respectively. A fifth mutant class was of pleiotropic nature and the sixth class comprised a Chlb-deficient mutant. Several of the mutants showed severe deficiencies in the levels of subunits of PS I, PS II or the cytochromeb6/f complex. Thus the mutational lesions could be located precisely. Only one mutant was defective in the transcript patterns of some plastid-encoded photosynthesis genes. Hence most of the mutants isolated appear to be affected in translational and post-translational regulatory processes of thylakoid membrane biogenesis or in structural genes encoding constituent subunits of the thylakoid protein complexes.

cis-Regulatory Elements for Mesophyll-Specific Gene Expression in the C4 Plant Flaveria trinervia, the Promoter of the C4 Phosphoenolpyruvate Carboxylase Gene

C4 photosynthesis depends on the strict compartmentalization of CO2 assimilatory enzymes. cis-regulatory mechanisms are described that ensure mesophyll-specific expression of the gene encoding the C4 isoform of phosphoenolpyruvate carboxylase (ppcA1) of the C4 dicot Flaveria trinervia. To elucidate and understand the anatomy of the C4 ppcA1 promoter, detailed promoter/reporter gene studies were performed in the closely related C4 species F. bidentis, revealing that the C4 promoter contains two regions, a proximal segment up to −570 and a distal part from −1566 to −2141, which are necessary but also sufficient for high mesophyll-specific expression of the β-glucuronidase reporter gene. The distal region behaves as an enhancer-like expression module that can direct mesophyll-specific expression when inserted into the ppcA1 promoter of the C3 plant F. pringlei. Mesophyll expression determinants were restricted to a 41-bp segment, referred to as mesophyll expression module 1 (Mem1). Evolutionary and functional studies identified the tetranucleotide sequence CACT as a key component of Mem1.

An mRNA blueprint for C4 photosynthesis derived from comparative transcriptomics of closely related C3 and C4 species

C4 photosynthesis involves alterations to the biochemistry, cell biology, and development of leaves. Together, these modifications increase the efficiency of photosynthesis, and despite the apparent complexity of the pathway, it has evolved at least 45 times independently within the angiosperms. To provide insight into the extent to which gene expression is altered between C3 and C4 leaves, and to identify candidates associated with the C4 pathway, we used massively parallel mRNA sequencing of closely related C3 (Cleome spinosa) and C4 (Cleome gynandra) species. Gene annotation was facilitated by the phylogenetic proximity of Cleome and Arabidopsis (Arabidopsis thaliana). Up to 603 transcripts differ in abundance between these C3 and C4 leaves. These include 17 transcription factors, putative transport proteins, as well as genes that in Arabidopsis are implicated in chloroplast movement and expansion, plasmodesmatal connectivity, and cell wall modification. These are all characteristics known to alter in a C4 leaf but that previously had remained undefined at the molecular level. We also document large shifts in overall transcription profiles for selected functional classes. Our approach defines the extent to which transcript abundance in these C3 and C4 leaves differs, provides a blueprint for the NAD-malic enzyme C4 pathway operating in a dicotyledon, and furthermore identifies potential regulators. We anticipate that comparative transcriptomics of closely related species will provide deep insight into the evolution of other complex traits.

Localization of genes for coupling factor subunits on the spinach plastid chromosome

Summary1)Messenger RNA obtained from spinach cotyledons directs the synthesis of all five CF1 subunits in vitro in a rabbit reticulocyte translation system. The alpha, beta and epsilon subunit polypeptides were found as translation products from ptRNA and whole-cell poly A−-RNA. The gamma and delta subunits were synthesized from whole-cell poly A+-RNA as precursors of substantially greater molecular weight indicating that they originate in the nucleus and are imported into the chloroplast. High resolution electrophoresis, immunoprecipitation with antibodies against individual CF1 subunits (Nelson et al. 1980), and proteolytic peptide mapping were employed to identify the products.2)The genes for alpha, beta and epsilon subunits of CF1 were located by hybrid-selected translation with matrix-immobilized ptDNA fragments of known map position. The genes for all three CF1 subunit polypeptides are located in the large single-copy segment (cf. Herrmann et al. 1980b) of the circular ptDNA and each gene appears to be present once on the chromosome. The genes for the beta and epsilon subunits lie near each other in immediate vicinity to the structural gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase. The gene for the alpha subunit is separated by approximately 40 kbp from this gene cluster, and located near the gene for the 32 kd photosystem II polypeptide (Driesel et al. 1980).3)Restriction fragments of spinach ptDNA with CF1 subunit genes were cloned into pBR 322 and used to construct detailed maps.

Biogenesis and origin of thylakoid membranes

Thylakoids are photosynthetically active membranes found in Cyanobacteria and chloroplasts. It is likely that they originated in photosynthetic bacteria, probably in close connection to the occurrence of photosystem II and oxygenic photosynthesis. In higher plants, chloroplasts develop from undifferentiated proplastids. These contain very few internal membranes and the whole thylakoid membrane system is built when chloroplast differentiation takes place. During cell and organelle division a constant synthesis of new thylakoid membrane material is required. Also, rapid adaptation to changes in light conditions and long term adaptation to a number of environmental factors are accomplished by changes in the lipid and protein content of the thylakoids. Thus regulation of synthesis and assembly of all these elements is required to ensure optimal function of these membranes.

A plastidial sodium-dependent pyruvate transporter

Pyruvate serves as a metabolic precursor for many plastid-localized biosynthetic pathways, such as those for fatty acids, terpenoids and branched-chain amino acids. In spite of the importance of pyruvate uptake into plastids (organelles within cells of plants and algae), the molecular mechanisms of this uptake have not yet been explored. This is mainly because pyruvate is a relatively small compound that is able to passively permeate lipid bilayers, which precludes accurate measurement of pyruvate transport activity in reconstituted liposomes. Using differential transcriptome analyses of C3 and C4 plants of the genera Flaveria and Cleome, here we have identified a novel gene that is abundant in C4 species, named BASS2 (BILE ACID:SODIUM SYMPORTER FAMILY PROTEIN 2). The BASS2 protein is localized at the chloroplast envelope membrane, and is highly abundant in C4 plants that have the sodium-dependent pyruvate transporter. Recombinant BASS2 shows sodium-dependent pyruvate uptake activity. Sodium influx is balanced by a sodium:proton antiporter (NHD1), which was mimicked in recombinant Escherichia coli cells expressing both BASS2 and NHD1. Arabidopsis thaliana bass2 mutants lack pyruvate uptake into chloroplasts, which affects plastid-localized isopentenyl diphosphate synthesis, as evidenced by increased sensitivity of such mutants to mevastatin, an inhibitor of cytosolic isopentenyl diphosphate biosynthesis. We thus provide molecular evidence for a sodium-coupled metabolite transporter in plastid envelopes. Orthologues of BASS2 can be detected in all the genomes of land plants that have been characterized so far, thus indicating the widespread importance of sodium-coupled pyruvate import into plastids.