Early Light-inducible Proteins Research Articles

Phylogenetic analysis and seasonal cold acclimation‐associated expression of early light‐induced protein genes of Rhododendron catawbiense

The early light-induced proteins (ELIPs) are nuclear-encoded, light stress-induced proteins located in thylakoid membranes and related to light-harvesting Chl a/b-binding proteins. Recent evidence from physiological and genetic (mutant) studies supports a photoprotective function for ELIPs, particularly when green tissues are exposed to high light intensities at suboptimal temperatures. Broad-leaved evergreens belonging to genus Rhododendron are often exposed to a combination of low temperatures and high light in their natural habitat as the understory plants in deciduous forests and, therefore, are expected to employ photoprotective strategies during overwintering phase. Here we report analysis and characterization of previously identified ELIP expressed sequence tags (ESTs) from winter-collected Rhododendron catawbiense leaves. 5' or 3' rapid amplification of complementary DNA ends (RACEs) coupled with bioinformatic analyses were used to identify seven unique ELIPs from the 40 ESTs and were designated as RcELIP1-RcELIP7. Phylogenetic analysis revealed separate clustering of ELIP homologs from lower plants, monocots and eudicots (including RcELIPs) and further indicated an evolutionary divergence of ELIPs among angiosperms and gymnosperms. To gain insights into the cold acclimation (CA) physiology of rhododendrons, relative and absolute quantitative expression of RcELIPs was examined during seasonal CA of R. catawbiense leaves using real time reverse transcriptase-polymerase chain reaction. All seven RcELIPs were distinctly upregulated during the CA. It is postulated that RcELIPs expression constitutes an adaptive response to cold and high light in winter-adapted rhododendron leaves and perhaps plays a key role in the protection of photosynthetic apparatus from these stresses.

The light stress‐induced protein ELIP2 is a regulator of chlorophyll synthesis in Arabidopsis thaliana

The early light-induced proteins (ELIPs) belong to the multigenic family of pigment-binding light-harvesting complexes. ELIPs accumulate transiently and are believed to play a protective role in plants exposed to high levels of light. Constitutive expression of the ELIP2 gene in Arabidopsis resulted in a marked reduction of the pigment content of the chloroplasts, both in mature leaves and during greening of etiolated seedlings. The chlorophyll loss was associated with a decrease in the number of photosystems in the thylakoid membranes, but the photosystems present were fully assembled and functional. A detailed analysis of the chlorophyll-synthesizing pathway indicated that ELIP2 accumulation downregulated the level and activity of two important regulatory steps: 5-aminolevulinate synthesis and Mg-protoporphyrin IX (Mg-Proto IX) chelatase activity. The contents of glutamyl tRNA reductase and Mg chelatase subunits CHLH and CHLI were lowered in response to ELIP2 accumulation. In contrast, ferrochelatase activity was not affected and the inhibition of Heme synthesis was null or very moderate. As a result of reduced metabolic flow from 5-aminolevulinic acid, the steady state levels of various chlorophyll precursors (from protoporphyrin IX to protochlorophyllide) were strongly reduced in the ELIP2 overexpressors. Taken together, our results indicate that the physiological function of ELIPs could be related to the regulation of chlorophyll concentration in thylakoids. This seems to occur through an inhibition of the entire chlorophyll biosynthesis pathway from the initial precursor of tetrapyrroles, 5-aminolevulinic acid. We suggest that ELIPs work as chlorophyll sensors that modulate chlorophyll synthesis to prevent accumulation of free chlorophyll, and hence prevent photooxidative stress.

Photosynthetic capacity and light harvesting efficiency during the winter‐to‐spring transition in subalpine conifers

Some coniferous forest ecosystems undergo complete photosynthetic down-regulation in winter. The present study examined the influence of several environmental parameters on intrinsic, needle-level photosynthesis and photoprotection during the spring reactivation of photosynthesis in subalpine conifers. Maximal photosystem II (PSII) efficiency, photosynthetic capacity, and amounts of zeaxanthin and early light-inducible protein (Elip) family members were assessed in three subalpine conifer species over 3 years, and intensively during the 2003 winter-to-spring transition. During summers, maximal PSII efficiency remained high while intrinsic photosynthetic capacity varied depending on precipitation. During winters and the winter-to-spring transition, photosynthetic capacity and PSII efficiency were highly correlated and (during the spring transition) strongly influenced by air and soil temperature and liquid water availability. Decreases in the amount of Elip family members from winter through spring paralleled disengagement of sustained zeaxanthin-dependent photoprotection, although one of four anti-Elip antibody-reactive bands increased during spring. Intrinsic photosynthetic capacity and maximal PSII efficiency were highly responsive to day-to-day environmental changes during spring, indicating that multiple environmental signals are integrated to orchestrate the reactivation of photosynthesis from the inactive winter state to the active summer state.

Winter down‐regulation of intrinsic photosynthetic capacity coupled with up‐regulation of Elip‐like proteins and persistent energy dissipation in a subalpine forest

Overwintering, sun-exposed and photosynthetically inactive evergreens require powerful photoprotection. The goal of this study was to seasonally characterize photosynthesis and key proteins/components involved in electron transport and photoprotection. Maximal photosystem II (PSII) efficiency and photosynthetic capacity, amounts of zeaxanthin (Z), antheraxanthin (A), pheophytin and proteins (oxygen-evolving 33 kDa protein (OEC), PSII core protein D1 and subunit S (PsbS) protein, and members of the early light-inducible protein (Elip) family) were assessed in five conifer species at high altitude and in ponderosa pine (Pinus ponderosa) at moderate altitude during summer and winter. Relative to summer, winter down-regulation of photosynthetic capacity and loss of PSII efficiency at the high-altitude sites were paralleled by decreases in OEC, D1, and pheophytin; massive nocturnal retention of (Z + A) and up-regulation of two to four proteins cross-reactive with anti-Elip antibodies; and no change in PsbS amount. By contrast, ponderosa pine at moderate altitude exhibited no down-regulation of photosynthetic capacity, smaller depressions in PSII efficiency, and less up-regulation of Elip family members. These results support a function for members of the Elip family in the acclimation of sun-exposed needles that down-regulate photosynthesis during winter. A possible role in sustained photoprotection is considered.

Differential Expression and Localization of Early Light-Induced Proteins in Arabidopsis

The early light-induced proteins (Elips) in higher plants are nuclear-encoded, light stress-induced proteins located in thylakoid membranes and related to light-harvesting chlorophyll (LHC) a/b-binding proteins. A photoprotective function was proposed for Elips. Here we showed that after 2 h exposure of Arabidopsis (Arabidopsis thaliana) leaves to light stress Elip1 and Elip2 coisolate equally with monomeric (mLhcb) and trimeric (tLhcb) populations of the major LHC from photosystem II (PSII) as based on the Elip:Lhcb protein ratio. A longer exposure to light stress resulted in increased amounts of Elips in tLhcb as compared to mLhcb, due to a reduction of tLhcb amounts. We demonstrated further that the expression of Elip1 and Elip2 transcripts was differentially regulated in green leaves exposed to light stress. The accumulation of Elip1 transcripts and proteins increased almost linearly with increasing light intensities and correlated with the degree of photoinactivation and photodamage of PSII reaction centers. A stepwise accumulation of Elip2 was induced when 40% of PSII reaction centers became photodamaged. The differential expression of Elip1 and Elip2 occurred also in light stress-preadapted or senescent leaves exposed to light stress but there was a lack of correlation between transcript and protein accumulation. Also in this system the accumulation of Elip1 but not Elip2 correlated with the degree of PSII photodamage. Based on pigment analysis, measurements of PSII activity, and assays of the oxidation status of proteins we propose that the discrepancy between amounts of Elip transcripts and proteins in light stress-preadapted or senescent leaves is related to a presence of photoprotective anthocyanins or to lower chlorophyll availability, respectively.

Suppression of Both ELIP1 and ELIP2 in Arabidopsis Does Not Affect Tolerance to Photoinhibition and Photooxidative Stress

ELIPs (early light-induced proteins) are thylakoid proteins transiently induced during greening of etiolated seedlings and during exposure to high light stress conditions. This expression pattern suggests that these proteins may be involved in the protection of the photosynthetic apparatus against photooxidative damage. To test this hypothesis, we have generated Arabidopsis (Arabidopsis thaliana) mutant plants null for both elip genes (Elip1 and Elip2) and have analyzed their sensitivity to light during greening of seedlings and to high light and cold in mature plants. In particular, we have evaluated the extent of damage to photosystem II, the level of lipid peroxidation, the presence of uncoupled chlorophyll molecules, and the nonphotochemical quenching of excitation energy. The absence of ELIPs during greening at moderate light intensities slightly reduced the rate of chlorophyll accumulation but did not modify the extent of photoinhibition. In mature plants, the absence of ELIP1 and ELIP2 did not modify the sensitivity to photoinhibition and photooxidation or the ability to recover from light stress. This raises questions about the photoprotective function of these proteins. Moreover, no compensatory accumulation of other ELIP-like proteins (SEPs, OHPs) was found in the elip1/elip2 double mutant during high light stress. elip1/elip2 mutant plants show only a slight reduction in the chlorophyll content in mature leaves and greening seedlings and a lower zeaxanthin accumulation in high light conditions, suggesting that ELIPs could somehow affect the stability or synthesis of these pigments. On the basis of these results, we make a number of suggestions concerning the biological function of ELIPs.

High-throughput virus-induced gene-silencing approach to assess the functional relevance of a moisture stress-induced cDNA homologous to lea4

The abiotic stress-responsive cDNA database and their expression profiles suggest that stress genes are many and diverse. However, characterization and validation of their functional significance has been a constraint to assessing their role in imparting tolerance. Virus-induced gene silencing (VIGS) is a potential option for assessing the functional significance of stress genes. Here the effectiveness of VIGS to silence the expression of an ABA-responsive lea4 (late embryogenic abundant) gene involved in stress tolerance is documented. In the present study, low moisture-stress protocols were developed in such a way that the plants experienced the desired stress level when silencing the target stress gene using VIGS was at a maximum. The functional relevance of a groundnut (Arachis hypogaea) subtracted-stress cDNA clone putative lea4 was examined by VIGS in tomato. A 400 bp fragment of lea4 was cloned into tobacco rattle virus-based VIGS vector to trigger post-transcriptional gene silencing by Agrobacterium-mediated inoculation in tomato plants. In silenced plants only lea4 transcripts showed a substantial decline, whereas the expression of other known stress-responsive genes such as apx (ascorbate peroxidase) and elip (early light-induced protein) were unaltered. Under moderate moisture stress, the silenced plants showed enhanced susceptibility as measured by cell viability, superoxide radical activity, and cell osmotic adjustment. This approach illustrates the potential benefits of VIGS in identifying functional relevance of low moisture stress-responsive genes. It is also demonstrated that heterologous probes with a fairly high degree of homology to the native genes can be used to study the functional relevance of stress-responsive genes using VIGS.

Cloning, molecular characterization, and expression analysis of several red clover cDNAs

To begin gathering information regarding nucleotide sequence similarity between red clover genes and other plant species, especially the model legume Medicago truncatula, several random red clover cDNAs were sequenced. The analyzed cDNAs included genes encoding actin; several proteins involved in photosynthesis including PsaH, PsbR, PsbX, early light-induced protein (ELIP), ferredoxin, chlorophyll a/b binding protein; fructose-bisphosphate aldolase;chloroplastic superoxide dismutase; and GTP-binding protein typA. The gene set had a median sequence identity of 92% with their counterparts from M. truncatula, suggesting its available genomics tools can be applied to red clover. An expression analysis of the gene set in various red clover tissues indicates the genes show a wide range of expression patterns. Consequently, this set of cDNAs and associated data are proving useful as controls in molecular genetic experiments involving red clover. Key words: Red clover, Trifolium pratense, Medicago truncatula, forage legume, genomics, inquiry-based learning

A Chlorophyll a/b-binding Protein Homolog That Is Induced by Iron Deficiency Is Associated with Enlarged Photosystem I Units in the Eucaryotic Alga Dunaliella salina

Adaptation of the halotolerant alga Dunaliella salina to iron deprivation involves extensive changes of chloroplast morphology, photosynthetic activities, and induction of a major 45-kDa chloroplast protein termed Tidi. Partial amino acid sequencing of proteolytic peptides suggested that Tidi resembles chlorophyll a/b-binding proteins which compose light-harvesting antenna complexes (LHC) (Varsano, T., Kaftan, D., and Pick, U. (2003) J. Plant Nutr. 26, 2197-2210). Here we show that Tidi shares the highest amino acid sequence similarity with light-harvesting I chlorophyll a/b-binding proteins from higher plants but has an extended proline-rich N-terminal domain. The accumulation of Tidi is reversed by iron supplementation, and its level is inversely correlated with photosystem I (PS-I) reaction center proteins. In native gel electrophoresis, Tidi co-migrates with enlarged PS-I-LHC-I super-complexes. Single particle electron microscopy analysis revealed that PS-I units from iron-deficient cells are larger (31 and 37 nm in diameter) than PS-I units from control cells (22 nm). The 77 K chlorophyll fluorescence emission spectra of isolated complexes suggest that the Tidi-LHC-I antenna are functionally coupled to the reaction centers of PS-I. These findings indicate that Tidi acts as an accessory antenna of PS-I. The enlargement of PS-I antenna in algae and in cyanobacteria under iron deprivation suggests a common limitation that requires rebalancing of the energy distribution between the two photosystems.

Mutational and expression analysis of ELIP1 and ELIP2 in Arabidopsis thaliana

Plants exposed to photoinhibitory conditions respond by accumulation of the early light-induced proteins (ELIPs) with a potential photoprotective function. In Arabidopsis thaliana two genes (Elip1 and Elip2) encode for two ELIP proteins: evidence exists that the two genes are differentially regulated but their precise function is unclear. Mutants null for one or the other Elip gene can help in elucidating ELIPs role and here we describe the expression profile of ELIP1 and ELIP2, and the phenotype of such null mutants. Both ELIPs accumulate during greening of etiolated seedlings and in mature plants the transcripts fluctuate diurnally without protein accumulation. Steady-state transcript level of both genes increases in response to high light with transcription of Elip1 much more sensitive than that of Elip2 to increasing irradiation at 22 degrees C. At 4 degrees C instead Elip2 is strongly transcribed even at growing light. Furthermore, only ELIP1 accumulates under high light at 22 degrees C while both proteins accumulate at 4 degrees C. These results indicate the existence of a differential regulation of ELIPs expression in response to light or chilling stress with mechanisms active either at transcriptional and post-transcriptional level. Phenotypically, the mutants behave as the wild type as far as sensitivity to light- or light and cold-induced short-term photoinhibition, while both ELIPs are necessary to ensure a high rate of chlorophyll accumulation during deetiolation in continuous high light.

Comparative analysis of expressed sequence tags from cold-acclimated and non-acclimated leaves of Rhododendron catawbiense Michx

An expressed sequence tag (EST) analysis approach was undertaken to identify major genes involved in cold acclimation of Rhododendron, a broad-leaf, woody evergreen species. Two cDNA libraries were constructed, one from winter-collected (cold-acclimated, CA; leaf freezing tolerance -53 degrees C) leaves, and the other from summer-collected (non-acclimated, NA; leaf freezing tolerance -7 degrees C) leaves of field-grown Rhododendron catawbiense plants. A total of 862 5'-end high-quality ESTs were generated by sequencing cDNA clones from the two libraries (423 from CA and 439 from NA library). Only about 6.3% of assembled unique transcripts were shared between the libraries, suggesting remarkable differences in gene expression between CA and NA leaves. Analysis of the relative frequency at which specific cDNAs were picked from each library indicated that four genes or gene families were highly abundant in the CA library including early light-induced proteins (ELIP), dehydrins/late embryogenesis abundant proteins (LEA), cytochrome P450, and beta-amylase. Similarly, seven genes or gene families were highly abundant in the NA library and included chlorophyll a/b-binding protein, NADH dehydrogenase subunit I, plastidic aldolase, and serine:glyoxylate aminotransferase, among others. Northern blot analyses for seven selected abundant genes confirmed their preferential expression in either CA or NA leaf tissues. Our results suggest that osmotic regulation, desiccation tolerance, photoinhibition tolerance, and photosynthesis adjustment are some of the key components of cold adaptation in Rhododendron.

The early light-inducible protein (ELIP) gene is expressed during the chloroplast-to-chromoplast transition in ripening tomato fruit.

Chloroplast-to-chromoplast transitions during fruit ripening require massive transformation of the plastid internal membrane structure as the photosynthetic apparatus is disassembled. Early Light-Inducible Proteins (ELIPs) are known to accumulate in chloroplasts during thylakoid biogenesis and under stressful conditions. To determine if ELIP may also play a role in thylakoid disassembly during the chloroplast-to-chromoplast transition, ELIP mRNA expression was measured in tomato, Lycopersicon esculentum Mill. cv. Rutgers. An EST clone was identified in the Tomato Genome Project/Solanaceae Genomics Network database that has high sequence similarity with the amino acid sequence of Arabidopsis ELIP1 and ELIP2. It has complete identity in the two conserved regions of the protein. Genomic Southern blots indicate that the gene is a single copy in tomato. The genomic sequence shows the three-exon structure typical of ELIP sequences from other species. mRNA for this gene is barely detectable on northern blots from etiolated seedlings, but transiently accumulates to high levels 2 h after transfer to the light. Greenhouse-grown tomatoes were used to measure ELIP mRNA accumulation during fruit development and ripening. Tomato ELIP mRNA is detectable in all stages of fruit ripening, but is most abundant in the breaker/turning stage of development. A survey of tomato EST databases revealed that ELIP cDNA is also relatively abundant in developing flowers, which contain yellow chromoplasts. Combined, these results suggest that ELIP may play a newly-recognized role in the chloroplast-to-chromoplast transition process.

Cold acclimation induced genes of trifoliate orange (Poncirus trifoliata).

Commercial citrus varieties are sensitive to low temperature. Poncirus trifoliata is a close relative of Citrus species and has been widely used as a cold-hardy rootstock for citrus production in low-temperature environments. mRNA differential display-reverse transcription (DDRT)-PCR and quantitative relative-RT-PCR were used to study gene expression of P. trifoliata under a gradual cold-acclimation temperature regime. Eight up-regulated cDNA fragments were isolated and sequenced. These fragments showed high similarities at the amino acid level to the following genes with known functions: betaine/proline transporter, water channel protein, aldo-keto reductase, early light-induced protein, nitrate transporter, tetratricopeptide-repeat protein, F-box protein, and ribosomal protein L15. These cold-acclimation up-regulated genes in P. trifoliata are also regulated by osmotic and photo-oxidative signals in other plants.

High-Intensity-Light-Dependent and Transient Expression of New Genes Encoding Distant Relatives of Light-Harvesting Chlorophyll-a/b Proteins in Chlamydomonas reinhardtii

We identified four Lhc-like genes (Lhl) encoding proteins that are distant relatives of light-harvesting chlorophyll a/b-binding (LHC) proteins in the green alga Chlamydomonas reinhardtii. Their mRNA levels after transfer from low-intensity light to high-intensity light (HL) were examined and compared with those of Lhc genes encoding LHC proteins of PSII. The transfer caused a decrease in the mRNA level of Lhl3, a homolog of Arabidopsis thaliana Lil3, within 2 h, followed by gradual restoration depending on the intensity of HL. The response was similar to that of Lhc genes. In contrast, the mRNA levels of Lhl1, Lhl2 and Lhl4 significantly increased, reached a maximum within 1 h after the transfer and then rapidly returned to a low level. The intensity of HL little influenced the response of these genes. While the Lhl1 and Lhl2 proteins were homologs of early light-inducible protein (ELIP) and high-light-inducible protein (HLIP), respectively, Lhl4 encoded a novel protein. The HL-induced expression of Lhl4 was most prominent among the genes tested. Studies using various inhibitors indicate that the HL response is not mediated by the redox state of plastoquinone pool or reactive oxygen species, but required de novo protein synthesis in the cytosol.

Transcript profiling of salinity stress responses by large-scale expressed sequence tag analysis in Mesembryanthemum crystallinum

The common ice plant, Mesembryanthemum crystallinum, is a halophytic (salt-loving) member of the Aizoaceae, which switches from C 3 photosynthesis to Crassulacean acid metabolism (CAM) when exposed to salinity or water-deficit stress. CAM is a metabolic adaptation of photosynthetic carbon fixation that improves water use efficiency by shifting net CO 2 uptake to the night, thereby reducing transpirational water loss. To improve our understanding of the molecular genetic underpinnings and control mechanisms for Crassulacean acid metabolism (CAM) and other salinity stress response adaptations, a total of 9733 expressed sequence tags (ESTs) from cDNAs derived from leaf tissues of well-watered and salinity-stressed (0.5 M NaCl for 30 and 48 h) were characterized. Clustering and assembly of these ESTs resulted in the identification of a total of 3676 tentative unique gene sequences (1249 tentative consensus sequences and 2427 singleton ESTs) expressed in leaves of ice plant under unstressed and salinity stressed conditions. The same number (2782) of ESTs from each library (total=8346 ESTs) were randomly selected and analyzed to compare expression profiles among the control and salt stressed leaf tissues. EST frequencies for transcripts encoding CAM-related enzymes, pathogenesis-related, senescence-associated, cell death-related, and stress-related proteins such as heat shock proteins (HSPs), chaperones, early light-inducible proteins, ion homeostasis, antioxidative stress, detoxification, and biosynthetic enzymes for osmoprotectants increased 2–12-fold in cDNA libraries constructed from salt stressed plants. In contrast, the frequency of ESTs encoding light-harvesting and photosystem complexes and C 3 photosynthetic enzymes decreased 4-fold overall following salinity stress with transcripts for ribulose bisphosphate carboxylase/oxygenase (RuBisCO) subunits decreasing 7-fold. Moreover, stressed plants contained a higher percentage of ESTs encoding novel and/or functionally unknown proteins. The rapid discovery of both known and unknown genes related to stress responses in M. crystallinum demonstrates the great utility of EST analysis in unraveling the complex set of adaptive mechanisms contributing to water use efficiency (CAM) and salinity tolerance.

Assembly of pigment-protein complexes in carotenoid-deficient membranes of plastids from wheat seedlings treated with norflurazon.

The pyridazinone-type herbicide norflurazon SAN 9789 inhibiting the biosynthesis of long-chain carotenoids results in significant decrease in PS II core complexes and content of light-harvesting complex (LHC) polypeptides. At the same time, early light-induced proteins (ELIP) with molecular masses of 20.5-16.5 and 13.5 kD disappear in norflurazon-treated seedlings grown under intermittent (pulsed) light, confirming the hypothesis that they are carotenoid-binding proteins. Full disappearance of Chl a forms at 668, 676, and 690 nm and a sharp decrease in Chl b form at 648 nm in treated seedlings grown under 30 or 100 lx light intensity shows close contact of these forms with carotenoids in the thylakoid membrane. The band shift from 740 to 720 nm in the low-temperature fluorescence spectrum (77 K) suggests a disturbance of energy transfer from LHC to the Chl a form at 710-712 nm.

Induction of early light-inducible protein gene expression in Pisum sativum after exposure to low levels of UV-B irradiation and other environmental stresses.

Plants are constantly subjected to environmental changes and have developed various defence mechanisms to facilitate their continued existence. Pisum sativum plants were exposed to low levels of UV-B radiation and ELIP (early light-inducible proteins) mRNA, with a probable protective function, was rapidly and strongly induced during this type of stress. To our knowledge, this is the only photosynthetic gene that is up-regulated following exposure to UV-B, and this result has to be compared with studies predominantly reporting down-regulation by UV-B of genes encoding proteins localised in the plastid. The expression pattern of ELIP mRNA in pea was also investigated during salt, wounding and ozone stress. The transcript levels of ELIP were induced after the salt and wounding treatments but not during ozone fumigation.

A genome?s-eye view of the light-harvesting polypeptides of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is a valuable model system for defining the structure and function of polypeptides of the photosynthetic apparatus and the dynamic aspects of photosynthesis. Recently, a genome-wide analysis of cDNAs and a draft genome sequence that covers approximately 90% of the genome were made available, providing a clear picture of the composition of specific gene families, the relationships among the gene family members, and the location of each member on the genome. We used the available sequence information to analyze the extensive family of light-harvesting genes in C. reinhardtii. There are nine genes encoding polypeptides of the major light-harvesting complex of photosystem II, two genes encoding the minor light-harvesting polypeptides of photosystem II, and nine genes encoding polypeptides predicted to comprise the photosystem I light-harvesting complex. Furthermore, there are five genes encoding early light-induced proteins and two genes encoding LI818 polypeptides. A candidate for the PsbS gene has also been found in the raw genome sequence data (Niyogi, personal communication), although no genes encoding homologues of the Sep, or Hli polypeptides have been identified. In this manuscript, we identify and classify the family of light-harvesting polypeptides encoded on the C. reinhardtii genome. This is an important first step in designing specific genetic, biochemical, and physiological studies aimed at characterizing the composition, function, and regulation of the light-harvesting complexes.

Cloning and Expression Analysis of Salt Responsive Gene from Kandelia candel

Identification of gene expression patterns in mangroves grown under salinity will help to reveal the molecular mechanisms of salt tolerance. Here, 10 cDNAs of genes were isolated from Kandelia candel and identified by representational difference analysis of cDNA (cDNA RDA) under different NaCl concentrations. Of five genes expressed preferentially under salt condition, two were unknown, three were two kinds of low molecular mass heat-shock proteins (sHSPs) and ADP-ribosylation factor, respectively. The expressions of other five genes were repressed under NaCl stress, two encoded cyclophilins, three were tonoplast intrinsic protein, early light-induced protein and 60S ribosomal protein, respectively.

What affects mRNA levels in leaves of field-grown aspen? A study of developmental and environmental influences.

We have analyzed the abundance of mRNAs expressed from 11 nuclear genes in leaves of a free-growing aspen (Populus tremula) tree throughout the growing season. We used multivariate statistics to determine the influence of environmental factors (i.e. the weather before sampling) and developmental responses to seasonal changes at the mRNA level for each of these genes. The gene encoding a germin-like protein was only expressed early in the season, whereas the other tested genes were expressed throughout the season and showed mRNA variations on a day-to-day basis. For six of the genes, reliable models were found that described the mRNA level as a function of weather, but the leaf age was also important for all genes except one encoding an early light-inducible protein (which appeared to be regulated purely by environmental factors under these conditions). The results confirmed the importance of several environmental factors previously shown to regulate the genes, but we also detected a number of less obvious factors (such as the variation in weather parameters and the weather of the previous day) that correlated with the mRNA levels of individual genes. The study shows the power of multivariate statistical methods in analyzing gene regulation under field conditions.