Cloning and selection of carotenoid ketolase genes for the engineering of high-yield astaxanthin in plants

β-Carotene ketolase (BKT) catalyzes the rate-limiting steps for the biosynthesis of astaxanthin. Several bkt genes have been isolated and explored to modify plant carotenoids to astaxanthin with limited success. In this study, five algal BKT cDNAs were isolated and characterized for the engineering of high-yield astaxanthin in plants. The products of the cDNAs showed high similarity in sequence and enzymatic activity of converting β-carotene into canthaxanthin. However, the enzymes exhibited extremely different activities in converting zeaxanthin into astaxanthin. Chlamydomonas reinhardtii BKT showed the highest conversion rate (ca 85%), whereas, Neochloris wimmeri BKT exhibited very poor activity of ketolating zeaxanthin. Expression of C. reinhardtii BKT in tobacco led to a twofold increase of total carotenoids in the leaves with astaxanthin being the predominant. The bkt genes described here provide a valuable resource for metabolic engineering of plants as cell factories for astaxanthin production.     

Functional characterization of various algal carotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis

Extending the carotenoid pathway to astaxanthin in plants is of scientific and industrial interest. However, expression of a microbial β-carotene ketolase (BKT) that catalyses the formation of ketocarotenoids in transgenic plants typically results in low levels of astaxanthin. The low efficiency of BKTs in ketolating zeaxanthin to astaxanthin is proposed to be the major limitation for astaxanthin accumulation in engineered plants. To verify this hypothesis, several algal BKTs were functionally characterized using an Escherichia coli system and three BKTs were identified, with high (up to 85%), moderate (～38%), and low (～1%) conversion rate from zeaxanthin to astaxanthin from Chlamydomonas reinhardtii (CrBKT), Chlorella zofingiensis (CzBKT), and Haematococcus pluvialis (HpBKT3), respectively. Transgenic Arabidopsis thaliana expressing the CrBKT developed orange leaves which accumulated astaxanthin up to 2 mg g(-1) dry weight with a 1.8-fold increase in total carotenoids. In contrast, the expression of CzBKT resulted in much lower astaxanthin content (0.24 mg g(-1) dry weight), whereas HpBKT3 was unable to mediate synthesis of astaxanthin in A. thaliana. The none-native astaxanthin was found mostly in a free form integrated into the light-harvesting complexes of photosystem II in young leaves but in esterified forms in senescent leaves. The alteration of carotenoids did not affect chlorophyll content, plant growth, or development significantly. The astaxanthin-producing plants were more tolerant to high light as shown by reduced lipid peroxidation. This study advances a decisive step towards the utilization of plants for the production of high-value astaxanthin.     

Accumulation of theanderose in association with development of freezing tolerance in the moss Physcomitrella patens

Mosses are known to have the ability to develop high degrees of resistance to desiccation and freezing stress at cellular levels. However, underlying cellular mechanisms leading to the development of stress resistance in mosses are not understood. We previously showed that freezing tolerance in protonema cells of the moss Physcomitrella patens was rapidly increased by exogenous application of the stress hormone abscisic acid (ABA) [Minami, A., Nagao, M., Arakawa, K., Fujikawa, S., Takezawa, D., 2003a. Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes. J. Plant Physiol. 160, 475-483]. Herein it is shown that protonema cells with acquired freezing tolerance specifically accumulate low-molecular-weight soluble sugars. Analysis of the most abundant trisaccharide revealed that the cells accumulated theanderose (G6-alpha-glucosyl sucrose) in close association with enhancement of freezing tolerance by ABA treatment. The accumulation of theanderose was inhibited by cycloheximide, an inhibitor of nuclear-encoded protein synthesis, coinciding with a remarkable decrease in freezing tolerance. Furthermore, theanderose accumulation was promoted by cold acclimation and treatment with hyperosmotic solutes, both of which had been shown to enhance cellular freezing tolerance. These results reveal a novel role for theanderose, whose biological function has been obscure, in high freezing tolerance in moss cells.     

Sequence analysis of expressed sequence tags from an ABA-treated cDNA library identifies stress response genes in the moss Physcomitrella patens.

Partial cDNA sequencing was used to obtain 169 expressed sequence tags (ESTs) in the moss, Physcomitrella patens. The source of ESTs was a random cDNA library constructed from 7 day-old protonemata following treatment with 10(-4) M abscisic acid (ABA). Analysis of the ESTs identified 69% with homology to known sequences, 61% of which had significant homology to sequences of plant origin. More importantly, at least 11 ESTs had significant similarities to genes which are implicated in plant stress-responses, including responses which may involve ABA. These included a cDNA associated with desiccation tolerance, two heat shock protein genes, one cold acclimation protein cDNA and five others that may be involved in either oxidative or chemical stress or both, i.e., Zn/Cu-superoxide dismutase, NADPH protochlorophyllide oxidoreductase (PorB), selenium binding protein, glutathione peroxidase and glutathione S transferase. Analysis of codon usage between P. patens and seed plants indicated that although mosses and higher plants are to a large extent similar, minor variations also exists that may represent the distinctiveness of each group.     

Use of an inducible reporter gene system for the analysis of auxin distribution in the moss<Emphasis Type="Italic"> Physcomitrella patens</Emphasis>

The plant hormone auxin plays a major role in a variety of growth and developmental responses, even in the more ancient plants-for example, cell differentiation in mosses. Nevertheless, almost nothing is known about the distribution of auxin during moss development. To address this question, we characterised auxin distribution in the moss Physcomitrella patens using auxin-inducible reporter gene systems. Stable transgenic Physcomitrella plants were produced expressing the beta-glucuronidase (GUS) gene driven by the auxin-inducible promoters GH3 and DR5, respectively. Both fusions showed remarkable differences with respect to auxin-induced promoter strength and expression kinetics. A detailed characterisation of the GUS expression pattern in different developmental stages revealed that the highest auxin concentrations were in dividing and ontogenetic young cells.     

Activation of the heat shock response in plants by chlorophenols: transgenic Physcomitrella patens as a sensitive biosensor for organic pollutants

The ability to detect early molecular responses to various chemicals is central to the understanding of biological impact of pollutants in a context of varying environmental cues. To monitor stress responses in a model plant, we used transgenic moss Physcomitrella patens expressing the beta-glucuronidase reporter (GUS) under the control of the stress-inducible promoter hsp17.3B. Following exposure to pollutants from the dye and paper industry, GUS activity was measured by monitoring a fluorescent product. Chlorophenols, heavy metals and sulphonated anthraquinones were found to specifically activate the hsp17.3B promoter (within hours) in correlation with long-term toxicity effects (within days). At mildly elevated physiological temperatures, the chemical activation of this promoter was strongly amplified, which considerably increased the sensitivity of the bioassay. Together with the activation of hsp17.3B promoter, chlorophenols induced endogenous chaperones that transiently protected a recombinant thermolabile luciferase (LUC) from severe heat denaturation. This sensitive bioassay provides an early warning molecular sensor to industrial pollutants under varying environments, in anticipation to long-term toxic effects in plants. Because of the strong cross-talk between abiotic and chemical stresses that we find, this P. patens line is more likely to serve as a direct toxicity bioassay for pollutants combined with environmental cues, than as an indicator of absolute toxicity thresholds for various pollutants. It is also a powerful tool to study the role of heat shock proteins (HSPs) in plants exposed to combined chemical and environmental stresses.     

Dual role for tomato heat shock protein 21: protecting photosystem II from oxidative stress and promoting color changes during fruit maturation

The tomato (Lycopersicon esculentum) chloroplast small heat shock protein (sHSP), HSP21, is induced by heat treatment in leaves, but also under normal growth conditions in developing fruits during the transition of chloroplasts to chromoplasts. We used transgenic tomato plants constitutively expressing HSP21 to study the role of the protein under stress conditions and during fruit maturation. Although we did not find any effect for the transgene on photosystem II (PSII) thermotolerance, our results show that the protein protects PSII from temperature-dependent oxidative stress. In addition, we found direct evidence of the protein's role in fruit reddening and the conversion of chloroplasts to chromoplasts. When plants were grown under normal growth temperature, transgenic fruits accumulated carotenoids earlier than controls. Furthermore, when detached mature green fruits were stored for 2 weeks at 2 degrees C and then transferred to room temperature, the natural accumulation of carotenoids was blocked. In a previous study, we showed that preheat treatment, which induces HSP21, allowed fruit color change at room temperature, after a cold treatment. Here, we show that mature green transgenic fruits constitutively expressing HSP21 do not require the heat treatment to maintain the ability to accumulate carotenoids after cold storage. This study demonstrates that a sHSP plays a role in plant development under normal growth conditions, in addition to its protective effect under stress conditions.     

小立碗藓（Physcomitrella patens）植物microRNA结合靶位预测

利用857条植物miRNA序列对27546条小立碗藓mRNA序列进行搜索,预测出162个植物miRNA家族在小立碗藓中存在结合靶位。miRNA结合靶位数目和miRNA协同作用网络分析结果同时显示,miR482和miR1168在小立碗藓中结合靶位多、协同作用广,提示它们对于小立碗藓可能具有重要生物学功能。52个菜茵衣藻特有的miRNA被预测在小立碗藓中存在结合靶位,显示小立碗藓在从藻类向种子植物进化过程中处在独特演化位置。     

Moss systems biology en route: phytohormones in Physcomitrella development

The moss Physcomitrella patens has become a powerful model system in modern plant biology. Highly standardized cell culture techniques, as well as the necessary tools for computational biology, functional genomics and proteomics have been established. Large EST collections are available and the complete moss genome will be released soon. A simple body plan and the small number of different cell types in Physcomitrella facilitate the study of developmental processes. In the filamentous juvenile moss tissue, developmental decisions rely on the differentiation of single cells. Developmental steps are controlled by distinct phytohormones and integration of environmental signals. Especially the phytohormones auxin, cytokinin, and abscisic acid have distinct effects on early moss development. In this article, we review current knowledge about phytohormone influences on early moss development in an attempt to fully unravel the complex regulatory signal transduction networks underlying the developmental decisions of single plant cells in a holistic systems biology approach.     

Is the High Basal Level of Salicylic Acid Important for Disease Resistance in Potato?

Potato (Solanum tuberosum) plants contain a high basal level of salicylic acid (SA), the role of which in disease resistance is currently unclear. Here we report that, in spite of a drastic reduction in total SA levels in transgenic potato plants expressing the bacterial salicylate hydroxylase gene (nahG), there was no significant increase in disease severity when infected by Phytophthora infestans. Therefore, the high basal level of SA does not lead to constitutive resistance in healthy potato plants. However, in contrast to control plants, arachidonic acid failed to induce systematic acquired resistance (SAR) in nahG plants against P. infestans, indicating an essential role of SA in potato SAR. These results suggest that in potato the development of SAR against P. infestans may involve increased sensitivity of the plant to SA.     

Regulatory mechanism controlling stomatal behavior conserved across 400 million years of land plant evolution

Stomatal pores evolved more than 410 million years ago [1,?2]?and allowed vascular plants to regulate transpirational water loss during the uptake of CO(2) for photosynthesis [3]. Here, we show that stomata on the sporophytes of the moss Physcomitrella patens [2] respond to environmental signals in a similar way to those of flowering plants [4] and that a homolog of a key signaling component in the vascular plant drought hormone abscisic acid (ABA) response [5] is?involved in stomatal control in mosses. Cross-species complementation experiments reveal that the stomatal ABA response of a flowering plant (Arabidopsis thaliana) mutant, lacking the ABA-regulatory protein kinase OPEN STOMATA 1 (OST1) [6], is rescued by substitution with the moss P.?patens homolog, PpOST1-1, which evolved more than 400 million years earlier. We further demonstrate through the targeted knockout of the PpOST1-1 gene in P.?patens that its role in guard cell closure is conserved, with stomata of mutant mosses exhibiting a significantly attenuated ABA response. Our analyses indicate that core regulatory components involved in guard cell ABA signaling of flowering plants are operational in mosses and likely originated in the last common ancestor of these lineages more than 400 million years ago [7], prior to the evolution of ferns [8, 9].     

Activation of SnRK2 by Raf-like kinase ARK represents a primary mechanism of ABA and abiotic stress responses

The Raf-like protein kinase abscisic acid (ABA) and abiotic stress-responsive Raf-like kinase (ARK) previously identified in the moss Physcomitrium (Physcomitrella) patens acts as an upstream regulator of subgroup III SNF1-related protein kinase2 (SnRK2), the key regulator of ABA and abiotic stress responses. However, the mechanisms underlying activation of ARK by ABA and abiotic stress for the regulation of SnRK2, including the role of ABA receptor-associated group A PP2C (PP2C-A), are not understood. We identified Ser1029 as the phosphorylation site in the activation loop of ARK, which provided a possible mechanism for regulation of its activity. Analysis of transgenic P. patens ark lines expressing ARK-GFP with Ser1029-to-Ala mutation indicated that this replacement causes reductions in ABA-induced gene expression, stress tolerance, and SnRK2 activity. Immunoblot analysis using an anti-phosphopeptide antibody indicated that ABA treatments rapidly stimulate Ser1029 phosphorylation in the wild type (WT). The phosphorylation profile of Ser1029 in ABA-hypersensitive ppabi1 lacking protein phosphatase 2C-A (PP2C-A) was similar to that in the WT, whereas little Ser1029 phosphorylation was observed in ABA-insensitive ark missense mutant lines. Furthermore, newly isolated ppabi1 ark lines showed ABA-insensitive phenotypes similar to those of ark lines. Therefore, ARK is a primary activator of SnRK2, preceding negative regulation by PP2C-A in bryophytes, which provides a prototype mechanism for ABA and abiotic stress responses in plants.

Phosphorylation in the activation loop of the Raf-like kinase ARK is critical for SNF1-related protein kinase2 regulation during abscisic acid responses in the moss Physcomitrium (Physcomitrella) patens.     

Exploration and identification of Physcomitrella patens moss peptides

In the current study the isolation and identification of Physcomitrella patens (Hedw.) B.S.G. moss peptides are described. Physcomitrella patens moss is actively used in recent years as a model organism to study the biology of plants. Protoplasts, protonemata and gametophores of the moss are demonstrated for the first time to contain diverse small peptides. From gametophores was isolated and identified 58 peptides that are fragments of 14 proteins, and from protonemata - 49 peptides, fragments of 15 proteins. It was found that the protonemata and gametophores Ph. patens, which are the successive stages of development of this plant, significantly different from each other as a peptide composition and the spectrum of the precursor protein of identified peptides. Isolation of protoplasts of the enzymatic destruction of cell wall protonemata accompanied by massive degradation of intracellular proteins, many of whom are proteins of photosynthesis, which is a characteristic response of plants to stress the impact of environmental factors. A total of moss protoplasts were isolated and identified 323 peptides that are fragments of 79 proteins.     

苔藓植物小立碗藓，功能基因组学研究新的模式系统

苔藓植物具有相对简单的发育模式,单倍体的配子体在其生活史中占主导地位,作为研究植物生物学过程的模式系统具有诸多的优越性。苔藓植物小立碗藓能够高效地通过同源重组的方式将外源核酸整合到其核DNA,这就使得基因打靶在此物种中就像在小鼠胚胎干细胞和酵母中一样成为一个非常便利的技术。另外由于小立碗藓与高等植物在生物特征上有很大相似之处加之其有其他诸多优越性,它有望成为一个诱人的植物生物学和功能基因组学研究的模式系统。Abstract：The potential of moss as a model system to study plant biological process is associated with their relatively simple developmental pattern and the dominance of the haploid gametophyte in the life cycle. The moss Physcomitrella patens exhibits a very high rate of homologous recombination in its nuclear DNA, making gene targeting approaches in this plant as convenient as in yeast or in ES cells of mice. Sharing many biological features with higher plants and having many other advantages, the moss Physcomitrella patens will be an attractive model system for plant biology and functional genome analysis.