Isolation and expression analysis of proline metabolism-related genes in Chrysanthemum lavandulifolium

Proline plays a significant role in plant resistance to abiotic stresses, and its level is determined by a combination of synthesis, catabolism and transport. The primary proteins involved are Δ¹-pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (PDH) and proline transporter (ProT). To utilise proline metabolism to improve the stress resistance of Chrysanthemum × morifolium, we isolated two P5CS-homologous genes (ClP5CS1 and ClP5CS2), one PDH gene (ClPDH) and four ProT-homologous genes (ClProT1-4) (GenBANK accession numbers: KF743136–KF743142) from Chrysanthemum lavandulifolium, which is closely related to chrysanthemums and exhibits strong resistance to stresses. Expression analysis of these genes in different organs and under various stresses indicated that ClP5CSs showed substantial constitutive expression, while ClPDH was only strongly expressed in the capitulum and was inhibited under most stresses. The expression patterns of four ClProT genes presented characteristics of organ specificity and disparity under stresses. Above all, the expression of ClProT2 was restricted to above-ground organs, especially strong in the capitulum and could be obviously induced by various stress conditions. Promoters of ClPDH and ClProTs contained many cis-acting regulatory elements involved in stress responses and plant growth and development. High levels of free proline were found in flower buds, the capitulum under the non-stress condition and later periods of stress conditions except cold treatment. Interestingly, organ specificity and disparity also exist in the level of free proline under different stress conditions. Our study indicates that ClProTs play significant roles in proline accumulation and stress responses, and that ClProT2 could be used to genetically modify the stress resistance of chrysanthemums. In addition, proline metabolism might be closely related to plant flowering and floral development.     

Reciprocal regulation of Δ 1-pyrroline-5-carboxylate synthetase and proline dehydrogenase genes controls proline levels during and after osmotic stress in plants

Plants generally accumulate free proline under osmotic stress conditions. Upon removal of the osmotic stress, the proline levels return to normal. In order to understand the mechanisms involved in regulating the levels of proline, we cloned and characterized a proline dehydrogenase (PDH) cDNA from Arabidopsis thaliana (AtPDH). The 1745?bp cDNA contains a major open reading frame encoding a peptide of 499 amino acids. The deduced amino acid sequence has high homology with both Saccharomyces cerevisiae and Drosophila melanogaster proline oxidases and contains a putative mitochondrial targeting sequence. When expressed in yeast, the AtPDH cDNA complemented a yeast put1 mutation and exhibited proline oxidase activity. We also determined the free proline contents and the Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and PDH mRNA levels under different osmotic stress and recovery conditions. The results demonstrated that the removal of free proline during the recovery from salinity or dehydration stress involves an induction of the PDH gene while the activity of P5CS declines. The reciprocal regulation of P5CS and PDH genes appears to be a key mechanism in the control of the levels of proline during and after osmotic stress. The PDH gene was also significantly induced by exogenously applied proline. The induction of PDH by proline, however, was inhibited by salt stress.     

Three <Emphasis Type="Italic">P5CS</Emphasis> genes including a novel one from <Emphasis Type="Italic">Lilium regale</Emphasis> play distinct roles in osmotic,drought and salt stress tolerance

Proline accumulations in abiotically stressed plants is generally considered to benefit their stress tolerance. The Δ¹-Pyrroline-5-carboxylate synthetase (P5CS) gene family, which encodes the rate-limiting enzyme in proline biosynthesis pathway, usually contains two duplicated genes in most plants. However, three P5CS genes including LrP5CS1, LrP5CS2 as well as a third one, LrP5CS3, were isolated from Lilium regale. LrP5CS3 is highly identical to LrP5CS1 in amino acid sequences, indicating they could come from a paralogous duplication. The phylogenetic tree suggested that the duplication of LrP5CS occurred independently after the divergence of Liliales and commelinoids. The expression of LrP5CS1 was strongly induced in leaves and roots both under drought and salinity, while that of LrP5CS3 was upregulated more moderately. LrP5CS2 stayed almost constitutive under stress. LrP5CS1 exhibited the highest activity after expressed in E. coli. Overexpression of LrP5CS genes conferred enhanced osmotic, drought and salt tolerance on transgenic Arabidopsis without negative effects in unstressed condition. Under salt stress, lines LrP5CS2 accumulated fewer proline than others, and lines LrP5CS1 grew better in root elongation. The roots of lines LrP5CS3 grew better than all others under unstressed condition and osmotic stress. Our study suggests that the three LrP5CS genes play distinct roles respectively in proline accumulation and abiotic stress tolerance.     

Regulation of gene expression governing proline metabolism in <Emphasis Type="Italic">Thellungiella salsuginea</Emphasis> by NaCl and paraquat

Differential expression of the proline metabolism genes in Thellungiella salsuginea (Pall) E. Schulz was investigated under salinity (100 and 300 mM NaCl), upon the effect of paraquat (0.1 μM), and at their joint action. It was shown that, depending on the intensity of stress factor, expression of the P5CS1 gene was induced in the leaves (at 100 mM NaCl) or roots (at 300 mM NaCl). When the plants on control medium were treated with paraquat, the proline content changed only in the leaves. Time course of proline content in the leaves complied with the dynamic of P5CS1 gene expression, while expression of PDH gene essentially did not change. When the plants, which experienced salt stress, were treated with paraquat, the content of proline and the P5CS1 mRNA level increased even more. The obtained results suggest a complicated nature of signaling between the organs of the halophyte Th. salsuginea causing expression of the proline biosynthesis genes in the leaves and roots under the effect of salinity, paraquat, or upon their joint action. The proline catabolism in these plants was maintained essentially unchanged, which is probably related to the participation of proline and/or the products of its degradation in the pathways of other metabolite biosynthesis. We suggested that proline took part in ROS scavenging process and proline level was under strong control in Th. salsuginea.     

Expression of the Vigna aconitifolia P5CSF129A gene in transgenic pigeonpea enhances proline accumulation and salt tolerance

Abiotic stress is the major limiting factor of plant growth and crop yield which can be improved by osmoprotectants. Proline acts as an osmoprotectant and plays an important role in osmotic balancing, protection of sub-cellular structures, enzymes and in increasing cellular osmolarity that provide the turgor necessary for cell expansion under stress conditions. ?1-pyrroline-5-carboxylate synthetase (P5CS), a rate-limiting enzyme in proline biosynthesis which is known for conferring enhanced salt and drought stress is subjected to feedback inhibition by proline. Therefore, in the present study, we used a mutagenized version P5CSF129A of wild P5CS which is not subjected to feedback control. Efficient in vitro transformation of embryonic structures of pigeonpea (Cajanus cajan (L.) Millsp.) was obtained using Agrobacterium tumefaciens strain LBA4404 harbouring a modified binary vector pCAMBIA 1301 carrying the hptII gene for resistance to hygromycin sulphate, GUS reporter gene, encoding β-glucuronidase, and the Vigna aconitifolia P5CSF129A genes under a constitutive 35S promoter. Embryonic structures showed blue color when tested for GUS after first cycle of antibiotic selection. Integration of T-DNA into nuclear genome of transformed plants and its sexual transmission to the progeny of the transgenic plants are confirmed by PCR amplification of 340 bp hptII, 800 bp P5CSF129A fragments and Southern blot hybridization analysis. The resultant primary transgenic plants showed more proline accumulation than their non-transformed plants. Levels of proline were also elevated in T1 transgenic plants when grown in the presence of 200 mM NaCl. In addition to their enhanced growth performance, more chlorophyll and relative water content under high salinity, these plants also had lower levels of lipid peroxidation. This suggests that overproduction of proline might play an important role against salt shock and cellular integrity.     

木薯及其他植物P5CS基因进化及表达分析

该研究采用生物信息学的方法,从木薯等31个已完成基因组测序的植物中,共鉴定出84个吡咯啉-5-羧酸合成酶(P5CS)基因,并对其进行系统发育分析。结果表明:(1)P5CS在内含子长度上差别较大,而在氨基酸长度、外显子数目、等电点和分子量上差别不大。(2)由于发生基因重复,在大多数单子叶和双子叶植物中都有2个P5CS,而且在单子叶和双子叶植物中均发现P5CS1基因聚类在一组,而P5CS2基因聚类在另一组,支持P5CS1和P5CS2基因是独立起源,且该重复事件发生在单子叶和双子叶植物分化之前。(3)在某些植物(如蒺藜苜蓿、大豆等)中存在3~7个P5CS基因,表明在单子叶和双子叶植物分化之后P5CS基因又发生了多次重复事件,并将它们归纳为4种进化模式。(4)木薯中有2个P5CS基因,表达分析显示,MeP5CS1和MeP5CS2在叶片、叶柄、茎、须根和储藏根中均可检测到,其中MeP5CS1在叶片中表达较高,而MeP5CS2在茎和储藏根中表达较高。(5)干旱胁迫下,MeP5CS1仅在第一片完全展开叶中被显著诱导,而MeP5CS2在第一片完全展开叶和老叶中均能被显著诱导;低温胁迫下,MeP5CS1和MeP5CS2在不同组织中均能被显著诱导,但具有不同的表达模式。研究表明,木薯的MeP5CS1和MeP5CS2基因在转录水平受到干旱、低温等非生物胁迫的调控。     

Molecular evolution of plant P5CS gene involved in proline biosynthesis

The P5CS ({Delta} 1-Pyrroline–5-Carboxylate Synthetase) gene encodes for a bifunctional enzyme that catalyzes the rate limiting reaction in proline biosynthesis in living organisms. A wide range of multifunctional roles of proline have now been shown in stress defense. The proline biosynthetic genes, especially, P5CS is commonly used in metabolic engineering for proline overproduction conferring stress tolerance in plants. The gene is functionally well characterized at the molecular level, but there is more to learn about its evolutionary path in the plant kingdom, particularly the drive behind functional (osmoprotective and developmental) divergence of duplication of P5CS genes. In this review, we present the current understanding of the evolutionary trail of plant P5CS gene which plays a key role in stress tolerance.     

Genetic engineering of chickpea (Cicer arietinum L.) with the P5CSF129A gene for osmoregulation with implications on drought tolerance

Abiotic stresses including water deficit severely limits crop yields in the semi-arid tropics. In chickpea, annual losses of over 3.7 million tones have been estimated to be due to water deficit conditions alone. Therefore, major efforts are needed to improve its tolerance to water deficit, and genetic engineering approaches provide an increasing hope for this possibility. We have used transgenic technology for the introduction of an osmoregulatory gene P5CSF129A encoding the mutagenized Δ¹-pyrroline-5-carboxylate synthetase (P5CS) for the overproduction of proline. A total of 49 transgenic events of chickpea were produced with the 35S:P5CSF129A gene through Agrobacterium tumefaciens-mediated gene transfer through the use of axillary meristem explants. Eleven transgenic events that accumulated high proline (2–6 folds) were further evaluated in greenhouse experiments based on their transpiration efficiency (TE), photosynthetic activity, stomatal conductance, and root length under water stress. Almost all the transgenic events showed a decline in transpiration at lower values of the fraction of transpirable soil water (dryer soil), and extracted more water than their untransformed parents. The accumulation of proline in the selected events was more pronounced that increased significantly in the leaves when exposed to water stress. However, the overexpression of P5CSF129A gene resulted only in a modest increase in TE, thereby indicating that the enhanced proline had little bearing on the components of yield architecture that are significant in overcoming the negative effects of drought stress in chickpea.     

The relationship between proline content, the expression level of P5CS (Δ1-pyrroline-5-carboxylate synthetase), and drought tolerance in Tibetan hulless barley (Hordeum vulgare var. nudum)

Many plants accumulate proline (Pro) when suffered from drought; thus, the relationship between Pro accumulation and plant drought tolerance becomes an increasing concern. Pro is synthesized from either glutamine or ornithine, and the former pathway dominates under osmotic stress conditions. In this study, the dynamic accumulation of free Pro under drought stress in 10 genotypes of Tibetan hulless barley (Hordeum vulgare var. nudum) with water lose rate (WLR) of 0.3304 to 0.5839 g/(h g dry wt) was investigated. However, no correlation between Pro accumulation and drought tolerance was found. Furthermore, the barley stripe mosaic virus establisheding virus-induced gene silencing was employed to suppress the expression of the encoding gene Δ¹-Pyrroline-5-Carboxylate Synthetase (P5CS), which catalyzes the ratelimiting step of Glu pathway in Pro biosynthesis. By the quantitative real-time polymerase chain reaction, the decrease of the P5CS expression was found, and a consequent Pro degradation was also detected in P5CS-silenced plants. However, neither increased WLR of detached leaves nor decreased survival rate under drought stress was found compared with control plants. These results suggested that the repressed expression of P5CS and decreased content of free Pro may not interfere with the drought tolerance of Tibetan hulless barley.     

Differential responses of the enzymes involved in proline biosynthesis and degradation in drought tolerant and sensitive cotton genotypes during drought stress and recovery

The relative water content (RWC), free proline levels and the activities of enzymes involved in proline metabolism were studied in drought tolerant (Ca/H 680) and drought sensitive (Ca/H 148) genotypes of cotton (Gossypium hirsutum L.) during induction of water stress and posterior recovery. Water stress caused a significant increase in proline levels and P5CS activity in leaves of both tolerant and sensitive genotypes, whereas the activity of P5CR increased minimally and the activity of OAT remains unchanged. The activity of PDH decreased under drought stress in both the genotypes. The leaf of tolerant genotype maintained higher RWC, photosynthetic activity and proline levels, as well as higher P5CS and P5CR activities under water stress than that of drought sensitive genotype. The drought induced proline levels and activities of P5CS and P5CR declined and tend to be equal to their respective controls, during recovery, whereas the PDH activity tends to increase. These results indicate that induction of proline levels by up regulation of P5CS and down regulation of PDH may be involved in the development of drought tolerance in cotton.     

The evolution of pyrroline-5-carboxylate synthase in plants: a key enzyme in proline synthesis

Many plants synthesize and accumulate proline in response to osmotic stress conditions. A central enzyme in the proline biosynthesis is the bifunctional enzyme Δ¹-pyrroline-5-carboxylate synthase (P5CS) that includes two functional catalytic domains: the γ-glutamyl kinase and the glutamic-γ-semialdehyde dehydrogenase. This enzyme catalyzes the first two steps of the proline biosynthetic pathway and plays a central role in the regulation of this process in plants. To determine the evolutionary events that occurred in P5CS genes, partial sequences from four Neotropical trees were cloned and compared to those of other plant taxa. Molecular phylogenetic analysis indicated that P5CS duplication events have occurred several times following the emergence of flowering plants and at different frequencies throughout the evolution of monocots and dicots. Despite the high number of conserved residues in plant P5CS sequences, positive selection was observed at different regions of P5CS paralogous genes and also when dicots and monocots were contrasted.     

A novel Δ-pyrroline-5-carboxylate synthetase gene of Medicago truncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation

Proline accumulates in environmentally stressed plant cells including those of legume roots and nodules, but how its level is regulated is poorly understood. Δ¹-Pyrroline-5-carboxylate synthetase (P5CS), the committed-step enzyme of proline biosynthesis, is encoded by two duplicated genes in many plants. Here, we isolated MtP5CS3, a third gene, from Medicago truncatula, whose predicted polypeptide sequence is highly similar to those of previously isolated MtP5CS1 and MtP5CS2 except an extra amino-terminal segment. MtP5CS3 was strongly expressed under salinity and drought in shoots and nodulating roots, while MtP5CS1 was constitutive and MtP5CS2 induced by abscisic acid. Under salinity, MtP5CS3 promoter was more active than those of MtP5CS1 and MtP5CS2, as shown by GUS fusions. Translationally fused MtP5CS1-GFP was localized in the cytoplasm, whereas significant proportions of MtP5CS2-GFP and MtP5CS3-GFP were co-localized with rubisco small subunit protein-fused RFP in transformed hairy root cells. Under salinity, RNA silencing of MtP5CS1 or MtP5CS2 strongly induced MtP5CS3 expression, while that of MtP5CS3 decreased free proline content and nodule number. Consistently, Mtp5cs3, a loss-of-function mutant, accumulated much less proline, formed fewer nodules, and fixed nitrogen significantly less efficiently than the wild type under salinity. Thus, MtP5CS3 plays a critical role in regulating stress-induced proline accumulation during symbiotic nitrogen fixation.     

Effects of low nitrogen and drought stresses on proline synthesis of <Emphasis Type="Italic">Jatropha curcas</Emphasis> seedling

Proline is one of the most important osmoregulatory solutes subjected to osmotic stresses. In this study, low nitrogen supply suppressed the dry biomass, leaf area, and proline biosynthesis of the seedlings of the energy plant Jatropha curcas, which could grow in poor, dry soil. Low-nitrogen stress induced JcP5CS mRNA expression and decreased the activity of P5CS enzyme and the content of free proline in leaves of J. curcas seedlings. When the seedlings grown in low-nitrogen conditions were suddenly exposed to PEG-6000 (−1.6 MPa) stress, the expression of JcP5CS gene was highly induced, and both the activity of P5CS and the content of free proline increased and maintained at high levels to mitigate the impact of drought stresses. This may be one of the reasons why J. curcas could adapt to poor and drought conditions.