Structural insights into a unique PSI-LHCI-LHCII-Lhcb9 supercomplex from moss Physcomitrium patens.

Photosystem I (PSI) possesses a variable supramolecular organization among different photosynthetic organisms to adapt to different light environments. Mosses are evolutionary intermediates that diverged from aquatic green algae and evolved into land plants. The moss Physcomitrium patens (P. patens) has a light-harvesting complex (LHC) superfamily more diverse than those of green algae and higher plants. Here, we solved the structure of a PSI-LHCI-LHCII-Lhcb9 supercomplex from P. patens at 2.68 Å resolution using cryo-electron microscopy. This supercomplex contains one PSI-LHCI, one phosphorylated LHCII trimer, one moss-specific LHC protein, Lhcb9, and one additional LHCI belt with four Lhca subunits. The complete structure of PsaO was observed in the PSI core. One Lhcbm2 in the LHCII trimer interacts with PSI core through its phosphorylated N terminus, and Lhcb9 mediates assembly of the whole supercomplex. The complicated pigment arrangement provided important information for possible energy-transfer pathways from the peripheral antennae to the PSI core.

ADH2/GSNOR1 is a key player in limiting genotoxic damage mediated by formaldehyde and UV-B in Arabidopsis.

Maintenance of genome stability is an essential requirement for all living organisms. Formaldehyde and UV-B irradiation cause DNA damage and affect genome stability, growth and development, but the interplay between these two genotoxic factors is poorly understood in plants. We show that Arabidopsis adh2/gsnor1 mutant, which lacks alcohol dehydrogenase 2/S-nitrosoglutathione reductase 1 (ADH2/GSNOR1), are hypersensitive to low fluence UV-B irradiation or UV-B irradiation-mimetic chemicals. Although the ADH2/GSNOR1 enzyme can act on different substrates, notably on S-hydroxymethylglutathione (HMG) and S-nitrosoglutathione (GSNO), our study provides several lines of evidence that the sensitivity of gsnor1 to UV-B is caused mainly by UV-B-induced formaldehyde accumulation rather than other factors such as alteration of the GSNO concentration. Our results demonstrate an interplay between formaldehyde and UV-B that exacerbates genome instability, leading to severe DNA damage and impaired growth and development in Arabidopsis, and show that ADH2/GSNOR1 is a key player in combating these effects.

Antenna arrangement and energy-transfer pathways of PSI-LHCI from the moss Physcomitrella patens.

Plants harvest light energy utilized for photosynthesis by light-harvesting complex I and II (LHCI and LHCII) surrounding photosystem I and II (PSI and PSII), respectively. During the evolution of green plants, moss is at an evolutionarily intermediate position from aquatic photosynthetic organisms to land plants, being the first photosynthetic organisms that landed. Here, we report the structure of the PSI-LHCI supercomplex from the moss Physcomitrella patens (Pp) at 3.23 Å resolution solved by cryo-electron microscopy. Our structure revealed that four Lhca subunits are associated with the PSI core in an order of Lhca1-Lhca5-Lhca2-Lhca3. This number is much decreased from 8 to 10, the number of subunits in most green algal PSI-LHCI, but the same as those of land plants. Although Pp PSI-LHCI has a similar structure as PSI-LHCI of land plants, it has Lhca5, instead of Lhca4, in the second position of Lhca, and several differences were found in the arrangement of chlorophylls among green algal, moss, and land plant PSI-LHCI. One chlorophyll, PsaF-Chl 305, which is found in the moss PSI-LHCI, is located at the gap region between the two middle Lhca subunits and the PSI core, and therefore may make the excitation energy transfer from LHCI to the core more efficient than that of land plants. On the other hand, energy-transfer paths at the two side Lhca subunits are relatively conserved. These results provide a structural basis for unravelling the mechanisms of light-energy harvesting and transfer in the moss PSI-LHCI, as well as important clues on the changes of PSI-LHCI after landing.

SHB1/HY1 Alleviates Excess Boron Stress by Increasing BOR4 Expression Level and Maintaining Boron Homeostasis in Arabidopsis Roots.

Boron is an essential mineral nutrient for higher plant growth and development. However, excessive amounts of boron can be toxic. Here, we report on the characterization of an Arabidopsis mutant, shb1 (sensitive to high-level of boron 1), which exhibits hypersensitivity to excessive boron in roots. Positional cloning demonstrated that the shb1 mutant bears a point mutation in a gene encoding a heme oxygenase 1 (HO1) corresponding to the HY1 gene involved in photomorphogenesis. The transcription level of the SHB1/HY1 gene in roots is up-regulated under excessive boron stimulation. Either overexpressing SHB1/HY1 or applying the HO1 inducer hematin reduces boron accumulation in roots and confers high boron tolerance. Furthermore, carbon monoxide and bilirubin, catalytic products of HO1, partially rescue the boron toxicity-induced inhibition of primary root growth in shb1. Additionally, the mRNA level of BOR4, a boron efflux transporter, is reduced in shb1 roots with high levels of boron supplementation, and hematin cannot relieve the boron toxicity-induced root inhibition in bor4 mutants. Taken together, our study reveals that HO1 acts via its catalytic by-products to promote tolerance of excessive boron by up-regulating the transcription of the BOR4 gene and therefore promoting the exclusion of excessive boron in root cells.

Nitric oxide induces monosaccharide accumulation through enzyme S-nitrosylation.

Nitric oxide (NO) is extensively involved in various growth processes and stress responses in plants; however, the regulatory mechanism of NO-modulated cellular sugar metabolism is still largely unknown. Here, we report that NO significantly inhibited monosaccharide catabolism by modulating sugar metabolic enzymes through S-nitrosylation (mainly by oxidizing dihydrolipoamide, a cofactor of pyruvate dehydrogenase). These S-nitrosylation modifications led to a decrease in cellular glycolysis enzymes and ATP synthase activities as well as declines in the content of acetyl coenzyme A, ATP, ADP-glucose and UDP-glucose, which eventually caused polysaccharide-biosynthesis inhibition and monosaccharide accumulation. Plant developmental defects that were caused by high levels of NO included delayed flowering time, retarded root growth and reduced starch granule formation. These phenotypic defects could be mediated by sucrose supplementation, suggesting an essential role of NO-sugar cross-talks in plant growth and development. Our findings suggest that molecular manipulations could be used to improve fruit and vegetable sweetness.

Nitric oxide modifies root growth by S-nitrosylation of plastidial glyceraldehyde-3-phosphate dehydrogenase.

Nitric oxide (NO) plays an essential role in a myriad of physiological and pathological processes, but the molecular mechanism of the action and the corresponding direct targets have remained largely unknown. We used cellular, biochemical, and genetic approaches to decipher the potential role of NO in root growth in Arabidopsis thaliana. We specifically demonstrate that exogenous application of NO simulates the phenotype of NO overproducing mutant (nox1), displaying reduced root growth and meristem size. Using root specific cell marker lines, we show that the cell in the cortex layer are more sensitive to NO as they show enhanced size. Examination of total S-nitrosylated proteins showed higher levels in nox1 mutant than wild type. Using an in vitro assay we demonstrate that plastidial glyderaldehyde-3-phosphate dehydrogenase (GAPDH) is one of NO direct targets. The function of GAPDH in glycolysis provide a rational for S-nitrosylation of this enzyme and its subsequent reduced activity and ultimately reduced growth in roots. Indeed, the rescue of the root growth phenotype in nox1 by exogenous application of glycine and serine, the downstream products of plastidial GAPDH provide unequivocal evidence for mechanism of NO action through S-nitrosylation of key proteins, thereby delicately balancing growth and stress responses.

Tetrahydrofolate Modulates Floral Transition through Epigenetic Silencing.

Folates, termed from tetrahydrofolate (THF) and its derivatives, function as coenzymes in one-carbon transfer reactions and play a central role in synthesis of nucleotides and amino acids. Dysfunction of cellular folate metabolism leads to serious defects in plant development; however, the molecular mechanisms of folate-mediated cellular modifications and physiological responses in plants are still largely unclear. Here, we reported that THF controls flowering time by adjusting DNA methylation-regulated gene expression in Arabidopsis (Arabidopsis thaliana). Wild-type seedlings supplied with THF as well as the high endogenous THF content mutant dihydrofolate synthetase folypoly-Glu synthetase homolog B exhibited significant up-regulation of the flowering repressor of Flowering Wageningen and thereby delaying floral transition in a dose-dependent manner. Genome-wide transcripts and DNA methylation profiling revealed that THF reduces DNA methylation so as to manipulate gene expression activity. Moreover, in accompaniment with elevated cellular ratios between monoglutamylated and polyglutamylated folates under increased THF levels, the content of S-adenosylhomo-Cys, a competitive inhibitor of methyltransferases, was obviously higher, indicating that enhanced THF accumulation may disturb cellular homeostasis of the concerted reactions between folate polyglutamylation and folate-dependent DNA methylation. In addition, we found that the loss-of-function mutant of CG DNA methyltransferase MET1 displayed much less responsiveness to THF-associated flowering time alteration. Taken together, our studies revealed a novel regulatory role of THF on epigenetic silencing, which will shed lights on the understanding of interrelations in folate homeostasis, epigenetic variation, and flowering control in plants.

Cytokinins can act as suppressors of nitric oxide in Arabidopsis.

Maintaining nitric oxide (NO) homeostasis is essential for normal plant physiological processes. However, very little is known about the mechanisms of NO modulation in plants. Here, we report a unique mechanism for the catabolism of NO based on the reaction with the plant hormone cytokinin. We screened for NO-insensitive mutants in Arabidopsis and isolated two allelic lines, cnu1-1 and 1-2 (continuous NO-unstressed 1), that were identified as the previously reported altered meristem program 1 (amp1) and as having elevated levels of cytokinins. A double mutant of cnu1-2 and nitric oxide overexpression 1 (nox1) reduced the severity of the phenotypes ascribed to excess NO levels as did treating the nox1 line with trans-zeatin, the predominant form of cytokinin in Arabidopsis. We further showed that peroxinitrite, an active NO derivative, can react with zeatin in vitro, which together with the results in vivo suggests that cytokinins suppress the action of NO most likely through direct interaction between them, leading to the reduction of endogenous NO levels. These results provide insights into NO signaling and regulation of its bioactivity in plants.

Exploring the mechanism of Physcomitrella patens desiccation tolerance through a proteomic strategy.

The moss Physcomitrella patens has been shown to tolerate abiotic stresses, including salinity, cold, and desiccation. To better understand this plant's mechanism of desiccation tolerance, we have applied cellular and proteomic analyses. Gametophores were desiccated over 1 month to 10% of their original fresh weight. We report that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling, and microtubule depolymerization. Despite the severe desiccation, the membrane system maintains integrity. Through two-dimensional gel electrophoresis and image analysis, we identified 71 proteins as desiccation responsive. Following identification and functional categorization, we found that a majority of the desiccation-responsive proteins were involved in metabolism, cytoskeleton, defense, and signaling. Degradation of cytoskeletal proteins might result in cytoskeletal disassembly and consequent changes in the cell structure. Late embryogenesis abundant proteins and reactive oxygen species-scavenging enzymes are both prominently induced, and they might help to diminish the damage brought by desiccation.

Differentially expressed genes under cold acclimation in Physcomitrella patens.

Cold acclimation improves freezing tolerance in plants. In higher plants, many advances have been made toward identifying the signaling and regulatory pathways that direct the low-temperature stress response; however, similar insights have not yet been gained for simple nonvascular plants, such as bryophytes. To elucidate the pathways that regulate cold acclimation in bryophytes, we used two PCR-based differential screening techniques, cDNA amplified fragment length polymorphism (cDNA-AFLP) and suppression subtractive hybridization (SSH), to isolate 510 ESTs that are differentially expressed during cold acclimation in Physcomitrella patens. We used realtime RT-PCR to further analyze expression of 29 of these transcripts during cold acclimation. Our results show that cold acclimation in the bryophyte Physcomitrella patens is not only largely similar to higher plants but also displays distinct differences, suggests significant alteration during the evolution of land plants.

The involvement of NtFtsZ2-1 gene in the regulation of chloroplast division and expansion in tobacco.

Chloroplasts are a vital group of organelles of plants, yet the molecular mechanisms associated with their division remain poorly understood. Recent studies have revealed that the FtsZ protein, known as a key component in prokaryotic cell division, is involved in chloroplast division process. The NtFtsZ2-1 gene was isolated from Nicotiana tabacum by RT-PCR, and the sense and antisense expression plasmids were used to examine the function of NtFtsZ2-1 gene in transgenic tobacco. Light and confocal observations revealed that the normal chloroplast division process was severely disrupted in transgenic plants with enhanced or reduced expression of NtFtsZ2-1 gene. These chloroplasts were abnormally larger in size and fewer in number compared with that of the wild-type tobacco. But the total chloroplast plan area per mesophyll cell was conserved in sense, antisense and wild type tobaccos. Analyses of electron micrographs and chlorophyll content of different transgenic plants showed that constitutively enhancing or inhibiting the expression of NtFtsZ2-1 gene had no direct influence on the ultrastructure and photosynthetic ability of chloroplasts. Basing on these results, we suggest that NtFtsZ2-1 gene is involved in chloroplast division and expansion; the fluctuation of NtFtsZ2-1 expression level would alter normal chloroplast number and size in plant cells. In addition, the similarities of ultrastructure and photosynthetic ability of chloroplasts among sense, antisense and wild type tobaccos implies that a special mechanism regulate the relationship between chloroplast number and size to maximize photosynthetic rate.

Cloning and functional characterization of PpDBF1 gene encoding a DRE-binding transcription factor from Physcomitrella patens.

The dehydration-responsive element binding (DREB) transcription factors play central roles in regulating expression of stress-inducible genes under abiotic stresses. In the present work, PpDBF1 (Physcomitrella patens DRE-binding Factor1) containing a conserved AP2/ERF domain was isolated from the moss P. patens. Sequence comparison and phylogenetic analysis revealed that PpDBF1 belongs to the A-5 group of DREB transcription factor subfamily. The transcriptional activation activity and DNA-binding specificity of PpDBF1 were verified by yeast one-hybrid and electrophoretic mobility shift assay experiments, and its nuclear localization was demonstrated by particle biolisitics. PpDBF1 transcripts were accumulated under various abiotic stresses and phytohormones treatments in P. patens, and transgenic tobacco plants over-expressing PpDBF1 gained higher tolerance to salt, drought and cold stresses. These results suggest that PpDBF1 may play a role in P. patens as a DREB transcription factor, implying that similar regulating systems are conserved in moss and higher plants.

[Extracellular Ca2+ signaling: first messenger in animals and plants].

It is well known that calcium acts as a vital intracellular second messenger that governs a large array of cellular processes. However, the molecular identification of a receptor for extracellular Ca2+, the extracellular calcium-sensing receptor, has opened up the possibility that Ca2+might also function as a messenger outside the cell. In animals, the Ca2+ sensor is the well-characterized extracellular-Ca2+- sensing receptor (CaR), a G- protein -coupled receptor originally isolated from the parathyroid gland. In addition, other receptors, channels and membrane proteins are all sensitive to external Ca2+ fluctuations. Recently, Han et al have cloned a receptor protein for extracellular calcium in Arabidopsis, which plays a key role in Ca2+-induced stomatal closing. Thus, the cloning of these receptors has prompted the consideration of Ca2+ also functioning as a 'first messenger' in animals and plants.

[Blue light signaling in mosses].

Arabidopsis thaliana contains five identified blue light photoreceptors and at least one unidentified blue/UV-A light photoreceptor. Cryptochromes (CRY1 and CRY2) modulate photomorphological processes, flowering time, and circadian timing while phototrophins (PHOT1 and PHOT2) modulate phototropism, chloroplast movement, and stomatal opening. Flavins are the chromophores and absorb in the blue and UV-A range. Considerable information is known about the structure and mode of action of these photoreceptors. The moss Physcomitrella patens contains two identified cryptochromes (CRY1a and CRY1b) which modulate side branch formation and auxin metabolism. Blue light-induced chloroplast movement was mediated by four phototropins. Transduction of the blue/UV-A stimulus does involve calcium signaling in moss cells.

[Cloning and expression in E.coli of Chlamydomonas reinhardtii CrFtsZ3 gene].

FtsZ protein plays a key role in the division of bacteria and chloroplast. To investigate the evolution of the chloroplast dividing apparatus, cloning and molecular characterization of a second chloroplast division gene, CrFtsZ3, from Chlamydomonas reinhardtii is performed. As there are two ftsZ genes in Chlamydomonas reinhardtii, duplication and divergence of the ftsZ genes might occur in an early stage before the emergence of green algae during the course of plant evolution. Sequence analysis showed that CrFtsZ3 gene had significant sequence homology with other ftsZs. It encoded a precursor of 479 amino acid residues with a putative transit peptide in its N-terminal. To study the function of CrFtsZ3, a recombinant plasmid expressing the full length CrFtsZ3/EGFP fusion protein was constructed. By using IPTG inducing, overexpression of CrFtsZ3/EGFP in E.coli was achieved, and this overexpression blocked cell division and resulted in filament formation. GFP-derived fluorescence showed regularly spaced dots along the bacterial filaments. This suggests that CrFtsZ3 could still recognize the signals of cell division site in E.coli and could take part in the process of bacterial division.

[RNA-based transgene silencing: mechanism and applications].

RNA-based transgene silencing is a phenomenon that endogenous and exogenous mRNAs are degraded specifically directed by double-stranded RNA. Here we review the recent advances on its mechanism and summarize related proteins and expatiate their functions. Furthermore,we introduce the enormous potential of dsRNA as a tool in functional genomics research and practical biotechnology.