Janny L. Peters

PHOTOMORPHOGENETIC MUTANTS OF HIGHER PLANTS

List of abbreviationsPhotomorphogenesis of higher plants is a complex process resulting from the co-action of at least three different photoreceptors: phytochrome (P), blue light (BL)/UV-A photoreceptor (cryptochrome) and UV-B photoreceptor (Mohr, 1986). The possible existence of multiple photoreceptor types [e.g. light-labile [type I] and light-stable [type 111 P) adds to the complexity (Vierstra and Quail, 1986; Pratt and Cordonnier, 1987; Nagatani et al . , 1987)l. Since both these types have very similar absorption spectra it is impossible to say which is responsible for a particular response. In addition, there appear to be multiple working mechanisms of some photoreceptors, e.g. very low fluence response (VLFR), low fluence response (LFR) and high irradiance response (HIR) of P (Kronenberg and Kendrick, 1986). Genotypes (often as induced mutants) in which certain parts of the morphogenetic pathway are eliminated, provide the tools for further physiological analysis. Such genotypes will exhibit a photomorphogenesis different and often simpler than their wild type. The relevance of the deleted part in the mutant is directly indicated by its difference in response compared to its isogenic wild type. Mutants with other defects, such as chlorophyll (Chl) or carotenoid deficiency, can also be very useful in photomorphogenetic research. The available literature is reviewed and the potential of a genetic approach to photomorphogenesis is outlined.

High pigment mutants of tomato exhibit high sensitivity for phytochrome action

Summary Anthocyanin synthesis and hypocotyl growth have been studied in tomato ( Lycopersicon esculentum Mill.) seedlings of high pigment ( hp ) mutants, aurea ( au ) mutants deficient in the labile type of phytochrome, an auhp double mutant and wild types. The phytochrome controlled [red light (RL)/far-red light reversible] anthocyanin synthesis occurring in a 24 h dark period after a 12 h pretreatment with RL or blue light (BL) is similar in the hp mutant, whereas in the case of the wild type, pretreatment with BL is more effective than RL. When grown under continuous broad-band UV-A, BL and RL for 5 d the au and auhp double mutants only accumulate low levels of anthocyanin compared to the wild type and the hp mutant. Under these conditions the hp mutant accumulates the highest levels of anthocyanin, but the relative effectiveness of RL and BL in the hp mutant and the wild type is reversed (BL being more effective than RL in wild type, whereas in the hp mutant RL is more effective than BL). These results suggest that the hp mutation enables maximal anthocyanin synthesis to be achieved without activation of the BL photoreceptor. When grown for 7 d in a regime of 14 h white light/10 h darkness the activation of anthocyanin synthesis is reduced in the au and auhp mutants compared to the wild type and the hp mutant. After the same treatment, the hypocotyls of hp -mutant seedlings of each cultivar are similar in length to those of their corresponding wild types and those of the au and auhp mutants are longer. However, after 5 d continuous low fluence rate (3 μ-tmol m- 2 s- 1 ) UV-A, BL and RL, the hypocotyl growth of the hp mutant is inhibited more than the wild type. The auhp double mutant shows a small but significantly higher anthocyanin accumulation than the au mutant, as well as increased inhibition of hypocotyl growth. It is proposed that the hp mutation increases the sensitivity to the labile phytochrome pool.

Physiological characterization of a high-pigment mutant of tomato.

Abstract— A high‐pigment (hp) mutant, which shows exaggerated phytochrome responses and three other genotypes of Lycopersicon esculenrum Mill. cv. Ailsa Craig: the aurea (au) mutant deficient in the bulk light‐labile phytochrome (PI) pool, the au, hp double mutant, and their isogenic wild type, were used in this study. Measurements of phytochrome destruction in red light (R) revealed that the exaggerated responses of the hp mutant are not caused by a higher absolute phytochrome level or a reduced rate of phytochrome destruction. Fluence‐response relationships for anthocyanin synthesis after a blue‐light pretreatment were studied to test if the hp mutant conveys hypersensitivity to the far‐red light (FR)‐absorbing form of phytochrome (Pfr), i.e. the threshold of Pfr required to initiate the response is lower. The response range for the hp mutant and wild type was identical, although the former exhibited a 6‐fold larger response. Moreover, the kinetics of anthocyanin accumulation in continuous R were similar in the wild‐type and hp‐mutant seedlings, despite the latter accumulating 9‐fold more anthocyanin. Since the properties of phytochrome are the same, the hp mutation appears to affect the state of responsiveness amplification, i.e. the same amount of Pfr leads to a higher response in the hp mutant. We therefore propose that the hp mutation is associated with an amplification step in the phytochrome transduction chain. Escape experiments showed that the anthocyanin synthesis after different light pretreatments terminated with a R pulse was still 50% FR reversible after 4–6 h darkness, indicating that the Pfr pool regulating this response must be relatively stable. However, fluence‐rate response relationships for anthocyanin synthesis and hypocotyl growth induced by a 24‐h irradiation with 451, 539, 649, 693, 704 and 729 nm light showed no or a severely reduced response in the au and au, hp mutants, suggesting the importance of PI in these responses. We therefore propose that the capacity for anthocyanin synthesis (state of responsiveness amplification) could be established by PI, while the anthocyanin synthesis is actually photoregulated via a stable Pfr pool. The Hp gene product is proposed to be an inhibitor of the state of responsiveness amplification for responses controlled by this relatively stable Pfr species.

SLO2, a mitochondrial pentatricopeptide repeat protein affecting several RNA editing sites, is required for energy metabolism

Pentatricopeptide repeat (PPR) proteins belong to a family of approximately 450 members in Arabidopsis, of which few have been characterized. We identified loss of function alleles of SLO2, defective in a PPR protein belonging to the E+ subclass of the P-L-S subfamily. slo2 mutants are characterized by retarded leaf emergence, restricted root growth, and late flowering. This phenotype is enhanced in the absence of sucrose, suggesting a defect in energy metabolism. The slo2 growth retardation phenotypes are largely suppressed by supplying sugars or increasing light dosage or the concentration of CO₂. The SLO2 protein is localized in mitochondria. We identified four RNA editing defects and reduced editing at three sites in slo2 mutants. The resulting amino acid changes occur in four mitochondrial proteins belonging to complex I of the electron transport chain. Both the abundance and activity of complex I are highly reduced in the slo2 mutants, as well as the abundance of complexes III and IV. Moreover, ATP, NAD+, and sugar contents were much lower in the mutants. In contrast, the abundance of alternative oxidase was significantly enhanced. We propose that SLO2 is required for carbon energy balance in Arabidopsis by maintaining the abundance and/or activity of complexes I, III, and IV of the mitochondrial electron transport chain.

HAWAIIAN SKIRT: An F-Box Gene That Regulates Organ Fusion and Growth in Arabidopsis

A fast neutron-mutagenized population of Arabidopsis (Arabidopsis thaliana) Columbia-0 wild-type plants was screened for floral phenotypes and a novel mutant, termed hawaiian skirt (hws), was identified that failed to shed its reproductive organs. The mutation is the consequence of a 28 bp deletion that introduces a premature amber termination codon into the open reading frame of a putative F-box protein (At3g61590). The most striking anatomical characteristic of hws plants is seen in flowers where individual sepals are fused along the lower part of their margins. Crossing of the abscission marker, ProPGAZAT:β-glucuronidase, into the mutant reveals that while floral organs are retained it is not the consequence of a failure of abscission zone cells to differentiate. Anatomical analysis indicates that the fusion of sepal margins precludes shedding even though abscission, albeit delayed, does occur. Spatial and temporal characterization, using ProHWS:β-glucuronidase or ProHWS:green fluorescent protein fusions, has identified HWS expression to be restricted to the stele and lateral root cap, cotyledonary margins, tip of the stigma, pollen, abscission zones, and developing seeds. Comparative phenotypic analyses performed on the hws mutant, Columbia-0 wild type, and Pro35S:HWS ectopically expressing lines has revealed that loss of HWS results in greater growth of both aerial and below-ground organs while overexpressing the gene brings about a converse effect. These observations are consistent with HWS playing an important role in regulating plant growth and development.

PHOTOCONTROL OF ANTHOCYANIN SYNTHESIS IN TOMATO SEEDLINGS: A GENETIC APPROACH*

The photocontrol of anthocyanin synthesis in dark‐grown seedlings of tomato (Lycopersicon esculentum Mill.) has been studied in an aurea (au) mutant which is deficient in the labile type of phytochrome, a high pigment (hp) mutant which has the wild‐type level of phytochrome and the double mutant au/hp, as well as the wild type. The hp mutant demonstrates phytochrome control of anthocyanin synthesis in response to a single red light (RL) pulse, whereas there is no measurable response in the wild type and au mutant. After pretreatment with 12 h blue light (BL) the phytochrome regulation of anthocyanin synthesis is 10‐fold higher in the hp mutant than in the wild type, whilst no anthocyanin is detectable in the au mutant, thus suggesting that it is the labile pool of phytochrome which regulates anthocyanin synthesis. The au/hp double mutant exhibits a small (3% of that in the hp mutant) RL/far‐red light (FR)‐reversible regulation of anthocyanin synthesis following a BL pretreatment. It is proposed that the hp mutant is hypersensitive to the FR‐absorbing form of phytochrome (Pfr) and that this (hypersensitivity) establishes response to the low level of Pfl. (below detection limits in phytochrome assays) in the au/hp double mutant.

Response of light-grown wild-type and aurea-mutant tomato plants to end-of-day far-red light.

Abstract The effect of end-of-day (EOD) far-red (FR) light treatment on tomato (Lycopersicon esculentum Mill.) plants of the wild type (WT) and an aurea (au) mutant (which is deficient in the spectrophotometrically detectable light-labile phytochrome pool in etiolated seedlings) was studied. Both the WT and au mutant exhibit a quantitatively similar EOD-FR response (i.e. stimulation of elongation growth) indicating that phytochrome is functional in light-grown seedlings of the au mutant. However, no dramatic effects of EOD-FR light on leaf elongation, chlorophyll, carotenoid and soluble protein levels were observed in either the WT or au mutant. The au mutant contained less chlorophyll and more soluble protein per unit fresh weight than the WT. Although practically no anthocyanin could be detected in leaves of the au mutant, the WT showed a tenfold reduction in anthocyanin in response to EOD-FR light. The lower productivity of the au mutant compared with the WT results from its reduced leaf surface area, since its maximum rate of photosynthesis or photosynthetic efficiency appears to be slightly higher than that of the WT. Phytochrome was extracted and partially purified from light-grown plants of the WT and au mutant and was quantified by spectrophotometry. On a fresh weight basis the phytochrome content of au-mutant plants was 66% of that of WT plants. It is proposed that the light-labile phytochrome pool regulates the synthesis of the photosynthetic apparatus in light-grown plants and that the light-stable phytochrome pool functions in the EOD-FR elongation response, the latter pool being present and functional in the au mutant.

Arabidopsis constitutive photomorphogenic mutant, bls1, displays altered brassinosteroid response and sugar sensitivity

We have isolated an Arabidopsis mutant impaired in light- and brassinosteroid (BR) induced responses, as well as in sugar signalling. The bls1 (brassinosteroid, light and sugar1) mutant displays short hypocotyl, expanded cotyledons, and de-repression of light-regulated genes in young seedlings, and leaf differentiation and silique formation on prolonged growth in dark. In light, the bls1 mutant is dwarf and develops a short root, compact rosette, with reduced trichome number, and exhibits delayed bolting. The activity of the BR inducible TCH4 and auxin inducible SAUR promoters, fused with GUS gene, is also altered in seedlings harbouring bls1 mutant background. In addition, the bls1 mutant is hypersensitive to metabolizable sugars. The short hypocotyl phenotype in dark, short root phenotype in light and sugar hypersensitivity could be rescued with BR application. Moreover, the bls1 mutant also showed higher expression of a BR biosynthetic pathway gene CPD, which is known to be feedback-regulated by BR. Using a genome-wide AFLP mapping strategy, the bls1 mutant has been mapped to a 1.4 Mb region of chromosome 5. Since no other mutant with essentially a similar phenotype has been assigned to this region, we suggest that the bls1 mutant defines a novel locus involved in regulating endogenous BR levels, with possible ramifications in integrating light, hormone and sugar signalling.

Generation of a 3D indexed Petunia insertion database for reverse genetics

BLAST searchable databases containing insertion flanking sequences have revolutionized reverse genetics in plant research. The development of such databases has so far been limited to a small number of model species and normally requires extensive labour input. Here we describe a highly efficient and widely applicable method that we adapted to identify unique transposon-flanking genomic sequences in Petunia. The procedure is based on a multi-dimensional pooling strategy for the collection of DNA samples; up to thousands of different templates are amplified from each of the DNA pools separately, and knowledge of their source is safeguarded by the use of pool-specific (sample) identification tags in one of the amplification primers. All products are combined into a single sample that is subsequently used as a template for unidirectional pyrosequencing. Computational analysis of the clustered sequence output allows automatic assignment of sequences to individual DNA sources. We have amplified and analysed transposon-flanking sequences from a Petunia transposon insertion library of 1000 individuals. Using 30 DNA isolations, 70 PCR reactions and two GS20 sequencing runs, we were able to allocate around 10 000 transposon flanking sequences to specific plants in the library. These sequences have been organized in a database that can be BLAST-searched for insertions into genes of interest. As a proof of concept, we have performed an in silico screen for insertions into members of the NAM/NAC transcription factor family. All in silico-predicted transposon insertions into members of this family could be confirmed in planta.

Multi-Level Interactions Between Heat Shock Factors, Heat Shock Proteins, and the Redox System Regulate Acclimation to Heat.

High temperature has become a global concern because it seriously affects the growth and reproduction of plants. Exposure of plant cells to high temperatures result in cellular damage and can even lead to cell death. Part of the damage can be ascribed to the action of reactive oxygen species (ROS), which accumulate during abiotic stresses such as heat stress. ROS are toxic and can modify other biomacromolecules including membrane lipids, DNA, and proteins. In order to protect the cells, ROS scavenging is essential. In contrast with their inherent harms, ROS also function as signaling molecules, inducing stress tolerance mechanisms. This review examines the evidence for crosstalk between the classical heat stress response, which consists of heat shock factors (HSFs) and heat shock proteins (HSPs), with the ROS network at multiple levels in the heat response process. Heat stimulates HSF activity directly, but also indirectly via ROS. HSFs in turn stimulate the expression of HSP chaperones and also affect ROS scavenger gene expression. In the short term, HSFs repress expression of superoxide dismutase scavenger genes via induction of miRNA398, while they also activate scavenger gene expression and stabilize scavenger protein activity via HSP induction. We propose that these contrasting effects allow for the boosting of the heat stress response at the very onset of the stress, while preventing subsequent oxidative damage. The described model on HSFs, HSPs, ROS, and ROS scavenger interactions seems applicable to responses to stresses other than heat and may explain the phenomenon of crossacclimation.