Mutators and sex in bacteria: Conflict between adaptive strategies

Bacterial mutation rates can increase and produce genetic novelty, as shown by in vitro and in silico experiments. Despite the cost due to a heavy deleterious mutation load, mutator alleles, which increase the mutation rate, can spread in asexual populations during adaptation because they remain associated with the rare favorable mutations they generate. This indirect selection for a genetic system generating diversity (second-order selection) is expected to be highly sensitive to changes in the dynamics of adaptation. Here we show by a simulation approach that even rare genetic exchanges, such as bacterial conjugation or transformation, can dramatically reduce the selection of mutators. Moreover, drift or competition between the processes of mutation and recombination in the course of adaptation reveal how second-order selection is unable to optimize the rate of generation of novelty.

Oxygen Requirement and Inhibition of C4 Photosynthesis1 : An Analysis of C4 Plants Deficient in the C3 and C4 Cycles

The basis for O2 sensitivity of C4 photosynthesis was evaluated using a C4-cycle-limited mutant of Amaranthus edulis (a phosphoenolpyruvate carboxylase-deficient mutant), and a C3-cycle-limited transformant of Flaveria bidentis (an antisense ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] small subunit transformant). Data obtained with the C4-cycle-limited mutant showed that atmospheric levels of O2 (20 kPa) caused increased inhibition of photosynthesis as a result of higher levels of photorespiration. The optimal O2 partial pressure for photosynthesis was reduced from approximately 5 kPa O2 to 1 to 2 kPa O2, becoming similar to that of C3 plants. Therefore, the higher O2 requirement for optimal C4 photosynthesis is specifically associated with the C4 function. With the Rubisco-limited F. bidentis, there was less inhibition of photosynthesis by supraoptimal levels of O2 than in the wild type. When CO2 fixation by Rubisco is limited, an increase in the CO2 concentration in bundle-sheath cells via the C4 cycle may further reduce the oxygenase activity of Rubisco and decrease the inhibition of photosynthesis by high partial pressures of O2 while increasing CO2 leakage and overcycling of the C4 pathway. These results indicate that in C4 plants the investment in the C3 and C4 cycles must be balanced for maximum efficiency.

Molecular markers reveal cryptic sex in the human pathogen Coccidioides immitis.

Coccidioides immitis, cause of a recent epidemic of "Valley fever" in California, is typical of many eukaryotic microbes in that mating and meiosis have yet to be reported, but it is not clear whether sex is truly absent or just cryptic. To find out, we have undertaken a population genetic study using PCR amplification, screening for single-strand conformation polymorphisms, and direct DNA sequencing to find molecular markers with nucleotide-level resolution. Both population genetic and phylogenetic analyses indicate that C. immitis is almost completely recombining. To our knowledge, this study is the first to find molecular evidence for recombination in a fungus for which no sexual stage has yet been described. These results motivate a directed search for mating and meiosis and illustrate the utility of single-strand conformation polymorphism and sequencing with arbitrary primer pairs in molecular population genetics.

Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes

Flock house virus (FHV), a single-stranded RNA insect virus, has previously been reported to cross the kingdom barrier and replicate in barley protoplasts and in inoculated leaves of several plant species [Selling, B. H., Allison, R. F. & Kaesberg, P. (1990) Proc. Natl. Acad. Sci. USA 87, 434–438]. There was no systemic movement of FHV in plants. We tested the ability of movement proteins (MPs) of plant viruses to provide movement functions and cause systemic spread of FHV in plants. We compared the growth of FHV in leaves of nontransgenic and transgenic plants expressing the MP of tobacco mosaic virus or red clover necrotic mosaic virus (RCNMV). Both MPs mobilized cell-to-cell and systemic movement of FHV in Nicotiana benthamiana plants. The yield of FHV was more than 100-fold higher in the inoculated leaves of transgenic plants than in the inoculated leaves of nontransgenic plants. In addition, FHV accumulated in the noninoculated upper leaves of both MP-transgenic plants. RCNMV MP was more efficient in mobilizing FHV to noninoculated upper leaves. We also report here that FHV replicates in inoculated leaves of six additional plant species: alfalfa, Arabidopsis, Brassica, cucumber, maize, and rice. Our results demonstrate that plant viral MPs cause cell-to-cell and long-distance movement of an animal virus in plants and offer approaches to the study of the evolution of viruses and mechanisms governing mRNA trafficking in plants as well as to the development of promising vectors for transient expression of foreign genes in plants.

Effects of Xylem pH on Transpiration from Wild-Type and flacca Tomato Leaves1 : A Vital Role for Abscisic Acid in Preventing Excessive Water Loss Even from Well-Watered Plants

The pH of xylem sap from tomato (Lycopersicon esculentum) plants increased from pH 5.0 to 8.0 as the soil dried. Detached wild-type but not flacca leaves exhibited reduced transpiration rates when the artificial xylem sap (AS) pH was increased. When a well-watered concentration of abscisic acid (0.03 μm) was provided in the AS, the wild-type transpirational response to pH was restored to flacca leaves. Transpiration from flacca but not from wild-type leaves actually increased in some cases when the pH of the AS was increased from 6.75 to 7.75, demonstrating an absolute requirement for abscisic acid in preventing stomatal opening and excessive water loss from plants growing in many different environments.

Alteration of Hormone Levels in Transgenic Tobacco Plants Overexpressing the Rice Homeobox Gene OSH1

The rice (Oryza sativa L.) homeobox gene OSH1 causes morphological alterations when ectopically expressed in transgenic rice, Arabidopsis thaliana, and tobacco (Nicotiana tabacum L.) and is therefore believed to function as a morphological regulator gene. To determine the relationship between OSH1 expression and morphological alterations, we analyzed the changes in hormone levels in transgenic tobacco plants exhibiting abnormal morphology. Levels of the plant hormones indole-3-acetic acid, abscisic acid, gibberellin (GA), and cytokinin (zeatin and trans-zeatin [Z]) were measured in leaves of OSH1-transformed and wild-type tobacco. Altered plant morphology was found to correlate with changes in hormone levels. The more severe the alteration in phenotype of transgenic tobacco, the greater were the changes in endogenous hormone levels. Overall, GA1 and GA4 levels decreased and abscisic acid levels increased compared with wild-type plants. Moreover, in the transformants, Z (active form of cytokinin) levels were higher and the ratio of Z to Z riboside (inactive form) also increased. When GA3 was supplied to the shoot apex of transformants, internode extension was restored and normal leaf morphology was also partially restored. However, such GA3-treated plants still exhibited some morphological abnormalities compared with wild-type plants. Based on these data, we propose the hypothesis that OSH1 affects plant hormone metabolism either directly or indirectly and thereby causes changes in plant development.

Molecular signals required for type III secretion and translocation of the Xanthomonas campestris AvrBs2 protein to pepper plants

Strains of Xanthomonas campestris pv. vesicatoria (Xcv) carrying avrBs2 are specifically recognized by Bs2 pepper plants, resulting in localized cell death and plant resistance. Agrobacterium-mediated transient expression of the Xcv avrBs2 gene in plant cells results in Bs2-dependent cell death, indicating that the AvrBs2 protein alone is sufficient for the activation of disease resistance-mediated cell death in planta. We now provide evidence that AvrBs2 is secreted from Xcv and that secretion is type III (hrp) dependent. N- and C-terminal deletion analysis of AvrBs2 has identified the effector domain of AvrBs2 recognized by Bs2 pepper plants. By using a truncated Pseudomonas syringae AvrRpt2 effector reporter devoid of type III signal sequences, we have localized the minimal region of AvrBs2 required for type III secretion in Xcv. Furthermore, we have identified the region of AvrBs2 required for both type III secretion and translocation to host plants. The mapping of AvrBs2 sequences sufficient for type III delivery also revealed the presence of a potential mRNA secretion signal.

Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway

Certain plant viruses encode suppressors of posttranscriptional gene silencing (PTGS), an adaptive antiviral defense response that limits virus replication and spread. The tobacco etch potyvirus protein, helper component-proteinase (HC-Pro), suppresses PTGS of silenced transgenes. The effect of HC-Pro on different steps of the silencing pathway was analyzed by using both transient Agrobacterium tumefaciens-based delivery and transgenic systems. HC-Pro inactivated PTGS in plants containing a preexisting silenced β-glucuronidase (GUS) transgene. PTGS in this system was associated with both small RNA molecules (21–26 nt) corresponding to the 3′ proximal region of the transcribed GUS sequence and cytosine methylation of specific sites near the 3′ end of the GUS transgene. Introduction of HC-Pro into these plants resulted in loss of PTGS, loss of small RNAs, and partial loss of methylation. These results suggest that HC-Pro targets a PTGS maintenance (as opposed to an initiation or signaling) component at a point that affects accumulation of small RNAs and methylation of genomic DNA.

Plant growth in elevated CO2 alters mitochondrial number and chloroplast fine structure

With increasing interest in the effects of elevated atmospheric CO2 on plant growth and the global carbon balance, there is a need for greater understanding of how plants respond to variations in atmospheric partial pressure of CO2. Our research shows that elevated CO2 produces significant fine structural changes in major cellular organelles that appear to be an important component of the metabolic responses of plants to this global change. Nine species (representing seven plant families) in several experimental facilities with different CO2-dosing technologies were examined. Growth in elevated CO2 increased numbers of mitochondria per unit cell area by 1.3–2.4 times the number in control plants grown in lower CO2 and produced a statistically significant increase in the amount of chloroplast stroma (nonappressed) thylakoid membranes compared with those in lower CO2 treatments. There was no observable change in size of the mitochondria. However, in contrast to the CO2 effect on mitochondrial number, elevated CO2 promoted a decrease in the rate of mass-based dark respiration. These changes may reflect a major shift in plant metabolism and energy balance that may help to explain enhanced plant productivity in response to elevated atmospheric CO2 concentrations.

Heterogeneous transcription of an indoleacetic acid biosynthetic gene in Erwinia herbicola on plant surfaces

We investigated the spatial pattern of expression of ipdC, a plant inducible gene involved in indoleacetic acid biosynthesis in Erwinia herbicola, among individual cells on plants to gain a better understanding of the role of this phenotype in the epiphytic ecology of bacteria and the factors involved in the regulation of ipdC. Nonpathogenic E. herbicola strain 299R harboring a transcriptional fusion of ipdC to gfp was inoculated onto bean plants, recovered from individual leaves 48 h after inoculation, and subjected to fluorescence in situ hybridization using a 16S rRNA oligonucleotide probe specific to strain 299R. Epifluorescence images captured through a rhodamine filter were used to distinguish the 5carboxytetramethylrhodamine-labeled cells of strain 299R from other leaf microflora. Quantification of the green fluorescence intensity of individual cells by analysis of digital images revealed that about 65% of the 299R cells recovered from bean leaves had higher ipdC expression than in culture. Additionally, 10% of the cells exhibited much higher levels of green fluorescence than the median fluorescence intensity, indicating that they are more heterogeneous with respect to ipdC expression on plants than in culture. Examination of 299R cells in situ on leaf surfaces by confocal laser scanning microscopy after fluorescence in situ hybridization of cells on leaf samples showed that even cells that were in close proximity exhibited dramatically different green fluorescence intensities, and thus, were in a physical or chemical microenvironment that induced differential expression of ipdC.

Manipulation of in Vivo Sorbitol Production Alters Boron Uptake and Transport in Tobacco1

Recent evidence that some species can retranslocate boron as complexes with sugar alcohols in the phloem suggests a possible mechanism for enhancing boron efficiency. We investigated the relationship between sugar alcohol (sorbitol) content, boron uptake and distribution, and translocation of foliar-applied, isotopically enriched 10B in three lines of tobacco (Nicotiana tabacum) plants differing in sorbitol production. In tobacco line S11, transformed with sorbitol-6-phosphate dehydrogenase, the production of sorbitol was accompanied by an increase in the concentration of boron in plant tissues and an increased uptake of boron compared with either tobacco line A4, transformed with antisense orientation of sorbitol-6-phosphate dehydrogenase, or wild-type tobacco (line SR1, zero-sorbitol producer). Foliar application of 10B to mature leaves was translocated to the meristematic tissues only in line S11. These results demonstrate that the concentration of the boron-complexing sugar alcohol in the plant tissue has a significant effect on boron uptake and distribution in plants, whereas the translocation of the foliar-applied 10B from the mature leaves to the meristematic tissues verifies that boron is mobile in sorbitol-producing plants (S11) as we reported previously. This suggests that selection or transgenic generation of cultivars with an increased sugar alcohol content can result in increased boron uptake, with no apparent negative effects on short-term growth.

Feedback Control and Diurnal Regulation of Gibberellin 20-Oxidase Transcript Levels in Potato1

Tuber formation in potato (Solanum tuberosum) is promoted by short photoperiods and is inhibited by gibberellins (GAs). Endogenous levels of GA1 were shown to decrease in stolons and leaves of potato plants induced to tuberize, which suggests that photoperiodic regulation of GA biosynthesis may play a role in tuber induction. We report the isolation of three potato cDNA clones (StGA20ox1–3) encoding GA 20-oxidase, a key regulatory enzyme in the GA-biosynthetic pathway. Using northern analysis, we detected a differential pattern of tissue-specific expression of the mRNAs corresponding to these clones. StGA20ox mRNAs were also very abundant in leaves of the potato ga1 mutant, which is blocked in the 13-hydroxylation step, and were strongly down-regulated by gibberellic acid, suggesting a feedback regulation of these genes. In plants grown in short-day (inductive) conditions, levels of the StGA20ox transcripts in leaves fluctuated during a 24-h period, with a peak of accumulation observed about 4 h after the lights were turned off. Interruption of the night with a 30-min “night break” of light (noninductive conditions) did not have a marked effect on the levels of accumulation of the three GA 20-oxidase mRNAs during the day, but it induced a second peak of expression of StGA20ox1 and StGA20ox3 transcripts late in the night. This observation, together with the finding that StGA20ox1 mRNA is expressed at high levels in leaves, suggests that night-break induction of this gene might play a role in the control of tuberization by regulating endogenous levels of GAs in response to daylength conditions.

Plant biology in the future

In the beginning of modern plant biology, plant biologists followed a simple model for their science. This model included important branches of plant biology known then. Of course, plants had to be identified and classified first. Thus, there was much work on taxonomy, genetics, and physiology. Ecology and evolution were approached implicitly, rather than explicitly, through paleobotany, taxonomy, morphology, and historical geography. However, the burgeoning explosion of knowledge and great advances in molecular biology, e.g., to the extent that genes for specific traits can be added (or deleted) at will, have created a revolution in the study of plants. Genomics in agriculture has made it possible to address many important issues in crop production by the identification and manipulation of genes in crop plants. The current model of plant study differs from the previous one in that it places greater emphasis on developmental controls and on evolution by differential fitness. In a rapidly changing environment, the current model also explicitly considers the phenotypic variation among individuals on which selection operates. These are calls for the unity of science. In fact, the proponents of “Complexity Theory” think there are common algorithms describing all levels of organization, from atoms all the way to the structure of the universe, and that when these are discovered, the issue of scaling will be greatly simplified! Plant biology must seriously contribute to, among other things, meeting the nutritional needs of the human population. This challenge constitutes a key part of the backdrop against which future evolution will occur. Genetic engineering technologies are and will continue to be an important component of agriculture; however, we must consider the evolutionary implications of these new technologies. Meeting these demands requires drastic changes in the undergraduate curriculum. Students of biology should be trained in molecular, cellular, organismal, and ecosystem biology, including all living organisms.

Chlorophyll Synthesis in Dark-Grown Pine Primary Needles1

The pigment content of dark-grown primary needles of Pinus jeffreyi L. and Pinus sylvestris L. was determined by high-performance liquid chromatography. The state of protochlorophyllide a and of chlorophylls during dark growth were analyzed by in situ 77 K fluorescence spectroscopy. Both measurements unambiguously demonstrated that pine primary needles are able to synthesize chlorophyll in the dark. Norflurazon strongly inhibited both carotenoid and chlorophyll synthesis. Needles of plants treated with this inhibitor had low chlorophyll content, contained only traces of xanthophylls, and accumulated carotenoid precursors. The first form of chlorophyll detected in young pine needles grown in darkness had an emission maximum at 678 nm. Chlorophyll-protein complexes with in situ spectroscopic properties similar to those of fully green needles (685, 695, and 735 nm) later accumulated in untreated plants, whereas in norflurazon-treated plants the photosystem I emission at 735 nm was completely lacking. To better characterize the light-dependent chlorophyll biosynthetic pathway in pine needles, the 77 K fluorescence properties of in situ protochlorophyllide a spectral forms were studied. Photoactive and nonphotoactive protochlorophyllide a forms with emission properties similar to those reported for dark-grown angiosperms were found, but excitation spectra were substantially red shifted. Because of their lower chlorophyll content, norflurazon-treated plants were used to study the protochlorophyllide a photoreduction process triggered by one light flash. The first stable chlorophyllide photoproduct was a chlorophyllide a form emitting at 688 nm as in angiosperms. Further chlorophyllide a shifts usually observed in angiosperms were not detected. The rapid regeneration of photoactive protochlorophyllide a from nonphotoactive protochlorophyllide after one flash was demonstrated.