Search tips
Search criteria

Results 1-25 (1015522)

Clipboard (0)

Related Articles

1.  Requirement of proline synthesis during Arabidopsis reproductive development 
BMC Plant Biology  2012;12:191.
Gamete and embryo development are crucial for successful reproduction and seed set in plants, which is often the determining factor for crop yield. Proline accumulation was largely viewed as a specific reaction to overcome stress conditions, while recent studies suggested important functions of proline metabolism also in reproductive development. Both the level of free proline and proline metabolism were proposed to influence the transition to flowering, as well as pollen and embryo development.
In this study, we performed a detailed analysis of the contribution of individual proline biosynthetic enzymes to vegetative development and reproductive success in Arabidopsis. In contrast to previous reports, we found that pyrroline-5-carboxylate (P5C) synthetase 2 (P5CS2) is not essential for sexual reproduction although p5cs2 mutant plants were retarded in vegetative development and displayed reduced fertility under long-day conditions. Single mutant plants devoid of P5CS1 did not show any developmental defects. Simultaneous absence of both P5CS isoforms resulted in pollen sterility, while fertile egg cells could still be produced. Expression of P5C reductase (P5CR) was indispensable for embryo development but surprisingly not needed for pollen or egg cell fertility. The latter observation could be explained by an extreme stability of P5CR activity, which had a half-life time of greater than 3 weeks in vitro. Expression of P5CR-GFP under the control of the endogenous P5CR promoter was able to restore growth of homozygous p5cr mutant embryos. The analysis of P5CR-GFP-fluorescence in planta supported an exclusively cytoplasmatic localisation of P5CR.
Our results demonstrate that potential alternative pathways for proline synthesis or inter-generation transfer of proline are not sufficient to overcome a defect in proline biosynthesis from glutamate during pollen development. Proline biosynthesis through P5CS2 and P5CR is limiting for vegetative and reproductive development in Arabidopsis, whereas disruption of P5CS1 alone does not affect development of non-stressed plants.
PMCID: PMC3493334  PMID: 23062072
Proline metabolism; Gamete and embryo development; Enzyme stability; Subcellular localisation
2.  Functional characterization of an ornithine cyclodeaminase-like protein of Arabidopsis thaliana 
BMC Plant Biology  2013;13:182.
In plants, proline synthesis occurs by two enzymatic steps starting from glutamate as a precursor. Some bacteria, including bacteria such as Agrobacterium rhizogenes have an Ornithine Cyclodeaminase (OCD) which can synthesize proline in a single step by deamination of ornithine. In A. rhizogenes, OCD is one of the genes transferred to the plant genome during the transformation process and plants expressing A. rhizogenes OCD have developmental phenotypes. One nuclear encoded gene of Arabidopsis thaliana has recently been annotated as an OCD (OCD-like; referred to here as AtOCD) but nothing is known of its function. As proline metabolism contributes to tolerance of low water potential during drought, it is of interest to determine if AtOCD affects proline accumulation or low water potential tolerance.
Expression of AtOCD was induced by low water potential stress and by exogenous proline, but not by the putative substrate ornithine. The AtOCD protein was plastid localized. T-DNA mutants of atocd and AtOCD RNAi plants had approximately 15% higher proline accumulation at low water potential while p5cs1-4/atocd double mutants had 40% higher proline than p5cs1 at low water potential but no change in proline metabolism gene expression which could directly explain the higher proline level. AtOCD overexpression did not affect proline accumulation. Enzymatic assays with bacterially expressed AtOCD or AtOCD purified from AtOCD:Flag transgenic plants did not detect any activity using ornithine, proline or several other amino acids as substrates. Moreover, AtOCD mutant or over-expression lines had normal morphology and no difference in root elongation or flowering time, in contrast to previous report of transgenic plants expressing A. rhizogenes OCD. Metabolite analysis found few differences between AtOCD mutants and overexpression lines.
The Arabidopsis OCD-like protein (AtOCD) may not catalyze ornithine to proline conversion and this is consistent with observation that three residues critical for activity of bacterial OCDs are not conserved in AtOCD. AtOCD was, however, stress and proline induced and lack of AtOCD expression increased proline accumulation by an unknown mechanism which did not require expression of P5CS1, the main enzyme responsible for stress-induced proline synthesis from glutamate. The results suggest that AtOCD may have function distinct from bacterial OCDs.
PMCID: PMC3840593  PMID: 24237637
Ornithine cyclodeaminase; Proline; Drought; Arabidopsis thaliana
3.  Proline induces heat tolerance in chickpea (Cicer arietinum L.) plants by protecting vital enzymes of carbon and antioxidative metabolism 
Chickpea is a heat sensitive crop hence its potential yield is considerably reduced under high temperatures exceeding 35 °C. In the present study, we evaluated the efficacy of proline in countering the damage caused by heat stress to growth and to enzymes of carbon and antioxidative metabolism in chickpea. The chickpea seeds were raised without (control) and with proline (10 μM) at temperatures of 30/25 °C, 35/30 °C, 40/35 °C and 45/40 °C as day/ night (12 h/12 h) in a growth chamber. The shoot and root length at 40/35 °C decreased by 46 and 37 %, respectively over control while at 45/40 °C, a decrease of 63 and 47 %, respectively over control was observed. In the plants growing in the presence of 10 μM proline at 40/35 °C and 45/40 °C, the shoot length showed improvement of 32 and 53 %, respectively over untreated plants, while the root growth was improved by 22 and 26 %, respectively. The stress injury (as membrane damage) increased with elevation of temperatures while cellular respiration, chlorophyll content and relative leaf water content reduced as the temperature increased to 45/40 °C. The endogenous proline was elevated to 46 μmol g−1 dw at 40/35 °C but declined to 19 μmol g−1 dw in plants growing at 45/40 °C that was associated with considerable inhibition of growth at this temperature. The oxidative damage measured as malondialdehyde and hydrogen peroxide content increased manifolds in heat stressed plants coupled with inhibition in the activities of enzymatic (superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase) and levels of non-enzymatic (ascorbic acid, glutathione, proline) antioxidants. The enzymes associated with carbon fixation (RUBISCO), sucrose synthesis (sucrose phosphate synthase) and sucrose hydrolysis (invertase) were strongly inhibited at 45/40 °C. The plants growing in the presence of proline accumulated proline up to 63 μmol g−1 dw and showed less injury to membranes, had improved content of chlorophyll and water, especially at 45/40 °C. Additionally, the oxidative injury was significantly reduced coupled with elevated levels of enzymatic and non-enzymatic antioxidants. A significant improvement was also noticed in the activities of enzymes of carbon metabolism in proline-treated plants. We report here that proline imparts partial heat tolerance to chickpea’s growth by reducing the cellular injury and protection of some vital enzymes related to carbon and oxidative metabolism and exogenous application of proline appears to have a countering effect against elevated high temperatures on chickpea.
PMCID: PMC3550571  PMID: 23573011
Chickpea; Carbon fixation; Heat stress; Oxidative stress; Proline
4.  Role of proline under changing environments 
Plant Signaling & Behavior  2012;7(11):1456-1466.
When exposed to stressful conditions, plants accumulate an array of metabolites, particularly amino acids. Amino acids have traditionally been considered as precursors to and constituents of proteins, and play an important role in plant metabolism and development. A large body of data suggests a positive correlation between proline accumulation and plant stress. Proline, an amino acid, plays a highly beneficial role in plants exposed to various stress conditions. Besides acting as an excellent osmolyte, proline plays three major roles during stress, i.e., as a metal chelator, an antioxidative defense molecule and a signaling molecule. Review of the literature indicates that a stressful environment results in an overproduction of proline in plants which in turn imparts stress tolerance by maintaining cell turgor or osmotic balance; stabilizing membranes thereby preventing electrolyte leakage; and bringing concentrations of reactive oxygen species (ROS) within normal ranges, thus preventing oxidative burst in plants. Reports indicate enhanced stress tolerance when proline is supplied exogenously at low concentrations. However, some reports indicate toxic effects of proline when supplied exogenously at higher concentrations. In this article, we review and discuss the effects of exogenous proline on plants exposed to various abiotic stresses. Numerous examples of successful application of exogenous proline to improve stress tolerance are presented. The roles played by exogenous proline under varying environments have been critically examined and reviewed.
PMCID: PMC3548871  PMID: 22951402
abiotic stress; antioxidant system; proline
5.  Proline Metabolism and Its Implications for Plant-Environment Interaction 
Proline has long been known to accumulate in plants experiencing water limitation and this has driven studies of proline as a beneficial solute allowing plants to increase cellular osmolarity during water limitation. Proline metabolism also has roles in redox buffering and energy transfer and is involved in plant pathogen interaction and programmed cell death. Some of these unique roles of proline depend on the properties of proline itself, whereas others depend on the “proline cycle” of coordinated proline synthesis in the chloroplast and cytoplasm with proline catabolism in the mitochondria. The regulatory mechanisms controlling proline metabolism, intercellular and intracellular transport and connections of proline to other metabolic pathways are all important to the in vivo functions of proline metabolism. Connections of proline metabolism to the oxidative pentose phosphate pathway and glutamate-glutamine metabolism are of particular interest. The N-acetyl glutamate pathway can also produce ornithine and, potentially, proline but its role and activity are unclear. Use of model systems such as Arabidopsis thaliana to better understand both these long studied and newly emerging functions of proline can help in the design of next-generation experiments testing whether proline metabolism is a promising metabolic engineering target for improving stress resistance of economically important plants.
PMCID: PMC3244962  PMID: 22303265
6.  Proline metabolism and cancer: emerging links to glutamine and collagen 
Purpose of review
Proline metabolism impacts a number of regulatory targets in both animals and plants and is especially important in cancer. Glutamine, a related amino acid, is considered second in importance only to glucose as a substrate for tumors. But proline and glutamine are interconvertible and linked in their metabolism. In animals, proline and glutamine have specific regulatory functions and their respective physiologic sources. A comparison of the metabolism of proline and glutamine would help us understand the importance of these two nonessential amino acids in cancer metabolism.
Recent findings
The regulatory functions of proline metabolism proposed 3 decades ago have found relevance in many areas. For cancer, these functions play a role in apoptosis, autophagy and in response to nutrient and oxygen deprivation. Importantly, proline-derived reactive oxygen species served as a driving signal for reprogramming. This model has been applied by others to metabolic regulation for the insulin-prosurvival axis, induction of adipose triglyceride lipase for lipid metabolism and regulation of embryonic stem cell development. Of special interest, modulatory proteins such as parkinson protein 7 and oral cancer overexpressed 1 interact with pyrroline-5-carboxylate reductase, a critical component of the proline regulatory axis. Although the interconvertibility of proline and glutamine has been long established, recent findings showed that the proto-oncogene, cellular myelocytomatosis oncogene, upregulates glutamine utilization (glutaminase) and routes glutamate to proline biosynthesis (pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductases). Additionally, collagen, which contains large amounts of proline, may be metabolized to serve as a reservoir for proline. This metabolic relationship as well as the new regulatory targets of proline metabolism invites an elucidation of the differential effects of these nonessential amino acids and their production, storage and mobilization.
Mechanisms by which the proline regulatory axis modulates the cancer phenotype are being revealed. Proline can be synthesized from glutamine as well as derived from collagen degradation. The metabolism of proline serves as a source of energy during stress, provides signaling reactive oxygen species for epigenetic reprogramming and regulates redox homeostasis.
PMCID: PMC4255759  PMID: 25474014
apoptosis; autophagy; collagen; glutamine; metabolic stress
7.  A double-flowered variety of lesser periwinkle (Vinca minor fl. pl.) that has persisted in the wild for more than 160 years 
Annals of Botany  2011;107(9):1445-1452.
Background and Aims
Homeotic transitions are usually dismissed by population geneticists as credible modes of evolution due to their assumed negative impact on fitness. However, several lines of evidence suggest that such changes in organ identity have played an important role during the origin and subsequent evolution of the angiosperm flower. Better understanding of the performance of wild populations of floral homeotic varieties should help to clarify the evolutionary potential of homeotic mutants. Wild populations of plants with changes in floral symmetry, or with reproductive organs replacing perianth organs or sepals replacing petals have already been documented. However, although double-flowered varieties are quite popular as ornamental and garden plants, they are rarely found in the wild and, if they are, usually occur only as rare mutant individuals, probably because of their low fitness relative to the wild-type. We therefore investigated a double-flowered variety of lesser periwinkle, Vinca minor flore pleno (fl. pl.), that is reported to have existed in the wild for at least 160 years. To assess the merits of this plant as a new model system for investigations on the evolutionary potential of double-flowered varieties we explored the morphological details and distribution of the mutant phenotype.
The floral morphology of the double-flowered variety and of a nearby population of wild-type plants was investigated by means of visual inspection and light microscopy of flowers, the latter involving dissected or sectioned floral organs.
Key Results
The double-flowered variety was found in several patches covering dozens of square metres in a forest within the city limits of Jena (Germany). It appears to produce fewer flowers than the wild-type, and its flowers are purple rather than blue. Most sepals in the first floral whorl resemble those in the wild-type, although occasionally one sepal is broadened and twisted. The structure of second-whorl petals is very similar to that of the wild-type, but their number per flower is more variable. The double-flowered character is due to partial or complete transformation of stamens in the third whorl into petaloid organs. Occasionally, ‘flowers within flowers’ also develop on elongated pedicels in the double-flowered variety.
The flowers of V. minor fl. pl. show meristic as well as homeotic changes, and occasionally other developmental abnormalities such as mis-shaped sepals or loss of floral determinacy. V. minor fl. pl. thus adds to a growing list of natural floral homeotic varieties that have established persistent populations in the wild. Our case study documents that even mutant varieties that have reproductive organs partially transformed into perianth organs can persist in the wild for centuries. This finding makes it at least conceivable that even double-flowered varieties have the potential to establish new evolutionary lineages, and hence may contribute to macroevolutionary transitions and cladogenesis.
PMCID: PMC3108809  PMID: 21527418
Double-flowered variety; homeosis; lesser periwinkle; macroevolution; Vinca minor fl. pl
8.  Proline Mechanisms of Stress Survival 
Antioxidants & Redox Signaling  2013;19(9):998-1011.
Significance: The imino acid proline is utilized by different organisms to offset cellular imbalances caused by environmental stress. The wide use in nature of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. Understanding the mechanisms by which proline enhances abiotic/biotic stress response will facilitate agricultural crop research and improve human health. Recent Advances: It is now recognized that proline metabolism propels cellular signaling processes that promote cellular apoptosis or survival. Studies have shown that proline metabolism influences signaling pathways by increasing reactive oxygen species (ROS) formation in the mitochondria via the electron transport chain. Enhanced ROS production due to proline metabolism has been implicated in the hypersensitive response in plants, lifespan extension in worms, and apoptosis, tumor suppression, and cell survival in animals. Critical Issues: The ability of proline to influence disparate cellular outcomes may be governed by ROS levels generated in the mitochondria. Defining the threshold at which proline metabolic enzyme expression switches from inducing survival pathways to cellular apoptosis would provide molecular insights into cellular redox regulation by proline. Are ROS the only mediators of proline metabolic signaling or are other factors involved? Future Directions: New evidence suggests that proline biosynthesis enzymes interact with redox proteins such as thioredoxin. An important future pursuit will be to identify other interacting partners of proline metabolic enzymes to uncover novel regulatory and signaling networks of cellular stress response. Antioxid. Redox Signal. 19, 998–1011.
PMCID: PMC3763223  PMID: 23581681
9.  The Making of a Compound Inflorescence in Tomato and Related Nightshades 
PLoS Biology  2008;6(11):e288.
Variation in the branching of plant inflorescences determines flower number and, consequently, reproductive success and crop yield. Nightshade (Solanaceae) species are models for a widespread, yet poorly understood, program of eudicot growth, where short side branches are initiated upon floral termination. This “sympodial” program produces the few-flowered tomato inflorescence, but the classical mutants compound inflorescence (s) and anantha (an) are highly branched, and s bears hundreds of flowers. Here we show that S and AN, which encode a homeobox transcription factor and an F-box protein, respectively, control inflorescence architecture by promoting successive stages in the progression of an inflorescence meristem to floral specification. S and AN are sequentially expressed during this gradual phase transition, and the loss of either gene delays flower formation, resulting in additional branching. Independently arisen alleles of s account for inflorescence variation among domesticated tomatoes, and an stimulates branching in pepper plants that normally have solitary flowers. Our results suggest that variation of Solanaceae inflorescences is modulated through temporal changes in the acquisition of floral fate, providing a flexible evolutionary mechanism to elaborate sympodial inflorescence shoots.
Author Summary
Among the most distinguishing features of plants are the flower-bearing shoots, called inflorescences. Despite a solid understanding of flower development, the molecular mechanisms that control inflorescence architecture remain obscure. We have explored this question in tomato, where mutations in two genes, ANANTHA (AN) and COMPOUND INFLORESCENCE (S), transform the well-known tomato “vine” into a highly branched structure with hundreds of flowers. We find that AN encodes an F-box protein ortholog of a gene called UNUSUAL FLORAL ORGANS that controls the identity of floral organs (petals, sepals, and so on), whereas S encodes a transcription factor related to a gene called WUSCHEL HOMEOBOX 9 that is involved in patterning the embryo within the plant seed. (F-box proteins are known for marking other proteins for degradation, but they can also function in hormone regulation and transcriptional activation) Interestingly, these genes have little or no effect on branching in inflorescences that grow continuously (so-called “indeterminate” shoots), as in Arabidopsis. However, we find that transient sequential expression of S followed by AN promotes branch termination and flower formation in plants where meristem growth ends with inflorescence and flower production (“determinate” shoots). We show that mutant alleles of s dramatically increase branch and flower number and have probably been selected for by breeders during modern cultivation. Moreover, the single-flower inflorescence of pepper (a species related to tomato, within the same Solanaceae family) can be converted to a compound inflorescence upon mutating its AN ortholog. Our results suggest a new developmental mechanism whereby inflorescence elaboration can be controlled through temporal regulation of floral fate.
Plant flower production is largely determined by the number of inflorescences, the branches produced on flower stems. Two genes identified in tomato reveal a new phase transition that may explain the mechanism of evolution of compound inflorescences in the Solanaceae family.
PMCID: PMC2586368  PMID: 19018664
10.  Moderate Increase of Mean Daily Temperature Adversely Affects Fruit Set of Lycopersicon esculentum by Disrupting Specific Physiological Processes in Male Reproductive Development 
Annals of Botany  2006;97(5):731-738.
• Background and Aims Global warming is gaining significance as a threat to natural and managed ecosystems since temperature is one of the major environmental factors affecting plant productivity. Hence, the effects of moderate temperature increase on the growth and development of the tomato plant (Lycopersicon esculentum) were investigated.
• Methods Plants were grown at 32/26 °C as a moderately elevated temperature stress (METS) treatment or at 28/22 °C (day/night temperatures) as a control with natural light conditions. Vegetative growth and reproductive development as well as sugar content and metabolism, proline content and translocation in the androecium were investigated.
• Key Results METS did not cause a significant change in biomass, the number of flowers, or the number of pollen grains produced, but there was a significant decrease in the number of fruit set, pollen viability and the number of pollen grains released. Glucose and fructose contents in the androecium (i.e. all stamens from one flower) were generally higher in the control than METS, but sucrose was higher in METS. Coincidently, the mRNA transcript abundance of acid invertase in the androecium was decreased by METS. Proline contents in the androecium were almost the same in the control and METS, while the mRNA transcript level of proline transporter 1, which expresses specifically at the surface of microspores, was significantly decreased by METS.
• Conclusions The research indicated that failure of tomato fruit set under a moderately increased temperature above optimal is due to the disruption of sugar metabolism and proline translocation during the narrow window of male reproductive development.
PMCID: PMC2803419  PMID: 16497700
Lycopersicon esculentum; moderately elevated temperature stress; microsporogenesis; mean daily temperature; fruit set; pollen release; male reproductive development; tapetum; hexose; sucrose; acid invertase; proline transporter
11.  Proline and hydroxyproline metabolism: implications for animal and human nutrition 
Amino acids  2010;40(4):1053-1063.
Proline plays important roles in protein synthesis and structure, metabolism (particularly the synthesis of arginine, polyamines, and glutamate via pyrroline-5-carboxylate), and nutrition, as well as wound healing, antioxidative reactions, and immune responses. On a pergram basis, proline plus hydroxyproline are most abundant in collagen and milk proteins, and requirements of proline for whole-body protein synthesis are the greatest among all amino acids. Therefore, physiological needs for proline are particularly high during the life cycle. While most mammals (including humans and pigs) can synthesize proline from arginine and glutamine/glutamate, rates of endogenous synthesis are inadequate for neonates, birds, and fish. Thus, work with young pigs (a widely used animal model for studying infant nutrition) has shown that supplementing 0.0, 0.35, 0.7, 1.05, 1.4, and 2.1% proline to a proline-free chemically defined diet containing 0.48% arginine and 2% glutamate dose dependently improved daily growth rate and feed efficiency while reducing concentrations of urea in plasma. Additionally, maximal growth performance of chickens depended on at least 0.8% proline in the diet. Likewise, dietary supplementation with 0.07, 0.14, and 0.28% hydroxyproline (a metabolite of proline) to a plant protein-based diet enhanced weight gains of salmon. Based on its regulatory roles in cellular biochemistry, proline can be considered as a functional amino acid for mammalian, avian, and aquatic species. Further research is warranted to develop effective strategies of dietary supplementation with proline or hydroxyproline to benefit health, growth, and development of animals and humans.
PMCID: PMC3773366  PMID: 20697752
Proline; Nutrition; Biochemistry; Health; Growth
12.  Nitrogen availability impacts oilseed rape (Brassica napus L.) plant water status and proline production efficiency under water-limited conditions 
Planta  2012;236(2):659-676.
Large amounts of nitrogen (N) fertilizers are used in the production of oilseed rape. However, as low-input methods of crop management are introduced crops will need to withstand temporary N deficiency. In temperate areas, oilseed rape will also be affected by frequent drought periods. Here we evaluated the physiological and metabolic impact of nitrate limitation on the oilseed rape response to water deprivation. Different amounts of N fertilizer were applied to plants at the vegetative stage, which were then deprived of water and rehydrated. Both water and N depletion accelerated leaf senescence and reduced leaf development. N-deprived plants exhibited less pronounced symptoms of wilting during drought, probably because leaves were smaller and stomata were partially closed. Efficiency of proline production, a major stress-induced diversion of nitrogen metabolism, was assessed at different positions along the whole plant axis and related to leaf developmental stage and water status indices. Proline accumulation, preferentially in younger leaves, accounted for 25–85 % of the free amino acid pool. This was mainly due to a better capacity for proline synthesis in fully N-supplied plants whether they were subjected to drought or not, as deduced from the expression patterns of the proline metabolism BnP5CS and BnPDH genes. Although less proline accumulated in the oldest leaves, a significant amount was transported from senescing to emerging leaves. Moreover, during rehydration proline was readily recycled. Our results therefore suggest that proline plays a significant role in leaf N remobilization and in N use efficiency in oilseed rape.
Electronic supplementary material
The online version of this article (doi:10.1007/s00425-012-1636-8) contains supplementary material, which is available to authorized users.
PMCID: PMC3404282  PMID: 22526495
Brassica napus; Drought stress; Nitrogen supply; Proline metabolism; Source-sink relationship; Water status
13.  A Regulatory Network for Coordinated Flower Maturation 
PLoS Genetics  2012;8(2):e1002506.
For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs.
Author Summary
Perfect flowers have both male organs that produce and release pollen and female organs that make and harbor seeds. Flowers also often attract pollinators using visual or chemical signals. So that male, female, and pollinator attraction functions occur at the right time, flower organs must grow and mature in a coordinated fashion. In the model self-pollinating plant Arabidopsis, a transcriptional network regulates genes that ensure coordinated growth of different flower organs, as well as pollen release and gynoecium (female) competence to support pollination. This network also regulates nectary development and production of volatile chemicals that may attract or repel insects. We have studied growth, chemical signal levels, and gene expression in mutants affected in components of this network, in order to determine how flower growth is controlled. Several plant hormones act in a cascade that promotes flower maturation. Moreover, regulatory feedback loops affect the timing and extent of developmental steps. Positive feedbacks may ensure that the development of different flower organs is coordinated and rapid, whereas negative feedbacks may allow growth to cease once flowers have opened. Our results provide a framework to understand how flower opening and reproduction are coordinated in Arabidopsis and other flowering plants.
PMCID: PMC3276552  PMID: 22346763
14.  Genetic association mapping identifies single nucleotide polymorphisms in genes that affect abscisic acid levels in maize floral tissues during drought 
Journal of Experimental Botany  2010;62(2):701-716.
In maize, water stress at flowering causes loss of kernel set and productivity. While changes in the levels of sugars and abscisic acid (ABA) are thought to play a role in this stress response, the mechanistic basis and genes involved are not known. A candidate gene approach was used with association mapping to identify loci involved in accumulation of carbohydrates and ABA metabolites during stress. A panel of single nucleotide polymorphisms (SNPs) in genes from these metabolic pathways and in genes for reproductive development and stress response was used to genotype 350 tropical and subtropical maize inbred lines that were well watered or water stressed at flowering. Pre-pollination ears, silks, and leaves were analysed for sugars, starch, proline, ABA, ABA-glucose ester, and phaseic acid. ABA and sugar levels in silks and ears were negatively correlated with their growth. Association mapping with 1229 SNPs in 540 candidate genes identified an SNP in the maize homologue of the Arabidopsis MADS-box gene, PISTILLATA, which was significantly associated with phaseic acid in ears of well-watered plants, and an SNP in pyruvate dehydrogenase kinase, a key regulator of carbon flux into respiration, that was associated with silk sugar concentration. An SNP in an aldehyde oxidase gene was significantly associated with ABA levels in silks of water-stressed plants. Given the short range over which decay of linkage disequilibrium occurs in maize, the results indicate that allelic variation in these genes affects ABA and carbohydrate metabolism in floral tissues during drought.
PMCID: PMC3003815  PMID: 21084430
ASI; abscisic acid; association mapping; drought; flower set; kernel set
15.  Transcriptomic and metabolomic analysis of Yukon Thellungiella plants grown in cabinets and their natural habitat show phenotypic plasticity 
BMC Plant Biology  2012;12:175.
Thellungiella salsuginea is an important model plant due to its natural tolerance to abiotic stresses including salt, cold, and water deficits. Microarray and metabolite profiling have shown that Thellungiella undergoes stress-responsive changes in transcript and organic solute abundance when grown under controlled environmental conditions. However, few reports assess the capacity of plants to display stress-responsive traits in natural habitats where concurrent stresses are the norm.
To determine whether stress-responsive changes observed in cabinet-grown plants are recapitulated in the field, we analyzed leaf transcript and metabolic profiles of Thellungiella growing in its native Yukon habitat during two years of contrasting meteorological conditions. We found 673 genes showing differential expression between field and unstressed, chamber-grown plants. There were comparatively few overlaps between genes expressed under field and cabinet treatment-specific conditions. Only 20 of 99 drought-responsive genes were expressed both in the field during a year of low precipitation and in plants subjected to drought treatments in cabinets. There was also a general pattern of lower abundance among metabolites found in field plants relative to control or stress-treated plants in growth cabinets. Nutrient availability may explain some of the observed differences. For example, proline accumulated to high levels in cold and salt-stressed cabinet-grown plants but proline content was, by comparison, negligible in plants at a saline Yukon field site. We show that proline accumulated in a stress-responsive manner in Thellungiella plants salinized in growth cabinets and in salt-stressed seedlings when nitrogen was provided at 1.0 mM. In seedlings grown on 0.1 mM nitrogen medium, the proline content was low while carbohydrates increased. The relatively higher content of sugar-like compounds in field plants and seedlings on low nitrogen media suggests that Thellungiella shows metabolic plasticity in response to environmental stress and that resource availability can influence the expression of stress tolerance traits under field conditions.
Comparisons between Thellungiella plants responding to stress in cabinets and in their natural habitats showed differences but also overlap between transcript and metabolite profiles. The traits in common offer potential targets for improving crops that must respond appropriately to multiple, concurrent stresses.
PMCID: PMC3568734  PMID: 23025749
16.  Further evidence that a terminal drought tolerance QTL of pearl millet is associated with reduced salt uptake 
•Pearl millet drought tolerance QTL on linkage group 2 is also associated with salt tolerance at reproductive growth stage.•Drought sensitive parent H77/833-2 accumulated higher root Na+ compared to tolerant PRLT 2/89-33 under short term salinity.•Rate of Na+ accumulation was faster in roots of H77/833-2, whereas it was gradual in PRLT2/89-33 and the two QTL-NILs.•Na+ ions accumulated preferentially in the older leaves of PRLT2/89-33 and two QTL-NILs; no trend observed in H77/833-2.
Earlier, we established that a major drought tolerance QTL on linkage group 2 of pearl millet is also associated with reduced salt uptake and enhanced growth under salt stress. Present study was undertaken to re-assess the performance of drought tolerant (PRLT 2/89-33) and drought sensitive (H 77/833-2) parents along with two QTL-NILs (ICMR 01029 and ICMR 01040), under salinity stress specifically imposed during post-flowering growth stages when plants had developed their ion sinks in full. Time course changes in ionic accumulation and their compartmentalization in different plant parts was studied, specifically to monitor and capture changes conferred by the two alleles at this QTL, at small intervals. Amongst different plant parts, higher accumulation of toxic ion Na+ was recorded in roots. Further, the Na+ concentration in roots of the testcross hybrid of the drought-sensitive parent (H 77/833-2) reached its maximum at ECiw 15 dS m−1 within 24 h after salinity imposition, whereas it continued to increase with time in the testcross hybrids of the drought tolerant parent PRLT 2/89-33 as well as those of its QTL-NILs (ICMR 01029 and ICMR 01004) and reached at its maximum at 120 h stage. Comparison of differential distribution of toxic ions in individual leaves revealed that Na+ ions were not uniformly distributed in the leaves of the drought-tolerant parent and drought-tolerant QTL-NILs; but accumulated preferentially in the older leaves, whereas the hybrid of the drought-sensitive parent showed significantly higher Na+ concentration in all main stem leaves irrespective of their age. Dynamics of chlorophyll and proline concentration variation studied under salt stress at late flowering stages revealed a greater reduction, almost twice, in both leaf chlorophyll and proline concentrations in younger leaves in the hybrids of the sensitive parent as compared to the tolerant parent and QTL NILs. Imposition of salinity stress even at flowering stage affected the yield performance in pearl millet, wherein higher yield was recorded in drought tolerant parent and the two QTL-NILs compared to drought sensitive parent.
PMCID: PMC4003388  PMID: 24895469
Pearl millet; Drought and salt tolerance; Terminal drought tolerance; QTL-NILs; Ionic accumulation; Ionic compartmentation; ABA, abscisic acid; DAS, days after sowing; DT-QTL, drought tolerance QTL; DT-QTL-NILs, DT-QTL-near isogenic lines; ECiw, electrical conductivity of the irrigation water; LG 2, linkage group 2
17.  Transcriptional and metabolomic analysis of Ascophyllum nodosum mediated freezing tolerance in Arabidopsis thaliana 
BMC Genomics  2012;13:643.
We have previously shown that lipophilic components (LPC) of the brown seaweed Ascophyllum nodosum (ANE) improved freezing tolerance in Arabidopsis thaliana. However, the mechanism(s) of this induced freezing stress tolerance is largely unknown. Here, we investigated LPC induced changes in the transcriptome and metabolome of A. thaliana undergoing freezing stress.
Gene expression studies revealed that the accumulation of proline was mediated by an increase in the expression of the proline synthesis genes P5CS1 and P5CS2 and a marginal reduction in the expression of the proline dehydrogenase (ProDH) gene. Moreover, LPC application significantly increased the concentration of total soluble sugars in the cytosol in response to freezing stress. Arabidopsis sfr4 mutant plants, defective in the accumulation of free sugars, treated with LPC, exhibited freezing sensitivity similar to that of untreated controls. The 1H NMR metabolite profile of LPC-treated Arabidopsis plants exposed to freezing stress revealed a spectrum dominated by chemical shifts (δ) representing soluble sugars, sugar alcohols, organic acids and lipophilic components like fatty acids, as compared to control plants. Additionally, 2D NMR spectra suggested an increase in the degree of unsaturation of fatty acids in LPC treated plants under freezing stress. These results were supported by global transcriptome analysis. Transcriptome analysis revealed that LPC treatment altered the expression of 1113 genes (5%) in comparison with untreated plants. A total of 463 genes (2%) were up regulated while 650 genes (3%) were down regulated.
Taken together, the results of the experiments presented in this paper provide evidence to support LPC mediated freezing tolerance enhancement through a combination of the priming of plants for the increased accumulation of osmoprotectants and alteration of cellular fatty acid composition.
PMCID: PMC3560180  PMID: 23171218
Arabidopsis thaliana; Ascophyllum nodosum; Freezing tolerance; Chemical priming; Soluble sugars; Metabolite profiling; Microarray analysis
18.  Mycorrhizal-Mediated Lower Proline Accumulation in Poncirus trifoliata under Water Deficit Derives from the Integration of Inhibition of Proline Synthesis with Increase of Proline Degradation 
PLoS ONE  2013;8(11):e80568.
Proline accumulation was often correlated with drought tolerance of plants infected by arbuscular mycorrhizal fungi (AMF), whereas lower proline in some AM plants including citrus was also found under drought stress and the relevant mechanisms have not been fully elaborated. In this study proline accumulation and activity of key enzymes relative to proline biosynthesis (▵1-pyrroline-5-carboxylate synthetase, P5CS; ornithine-δ-aminotransferase, OAT) and degradation (proline dehydrogenase, ProDH) were determined in trifoliate orange (Poncirus trifoliata, a widely used citrus rootstock) inoculated with or without Funneliformis mosseae and under well-watered (WW) or water deficit (WD). AMF colonization significantly increased plant height, stem diameter, leaf number, root volume, biomass production of both leaves and roots and leaf relative water content, irrespectively of water status. Water deficit induced more tissue proline accumulation, in company with an increase of P5CS activity, but a decrease of OAT and ProDH activity, no matter whether under AM or no-AM. Compared with no-AM treatment, AM treatment resulted in lower proline concentration and content in leaf, root, and total plant under both WW and WD. The AMF colonization significantly decreased the activity of both P5CS and OAT in leaf, root, and total plant under WW and WD, except for an insignificant difference of root OAT under WD. The AMF inoculation also generally increased tissue ProDH activity under WW and WD. Plant proline content significantly positively correlated with plant P5CS activity, negatively with plant ProDH activity, but not with plant OAT activity. These results suggest that AM plants may suffer less from WD, thereby inducing lower proline accumulation, which derives from the integration of an inhibition of proline synthesis with an enhancement of proline degradation.
PMCID: PMC3832396  PMID: 24260421
19.  Alleviation of Cadmium Toxicity in Brassica juncea L. (Czern. & Coss.) by Calcium Application Involves Various Physiological and Biochemical Strategies 
PLoS ONE  2015;10(1):e0114571.
Calcium (Ca) plays important role in plant development and response to various environmental stresses. However, its involvement in mitigation of heavy metal stress in plants remains elusive. In this study, we examined the effect of Ca (50 mM) in controlling cadmium (Cd) uptake in mustard (Brassica juncea L.) plants exposed to toxic levels of Cd (200 mg L−1 and 300 mg L−1). The Cd treatment showed substantial decrease in plant height, root length, dry weight, pigments and protein content. Application of Ca improved the growth and biomass yield of the Cd-stressed mustard seedlings. More importantly, the oil content of mustard seeds of Cd-stressed plants was also enhanced with Ca treatment. Proline was significantly increased in mustard plants under Cd stress, and exogenously sprayed Ca was found to have a positive impact on proline content in Cd-stressed plants. Different concentrations of Cd increased lipid peroxidation but the application of Ca minimized it to appreciable level in Cd-treated plants. Excessive Cd treatment enhanced the activities of antioxidant enzymes superoxide dismutase, ascorbate peroxidase and glutathione reductase, which were further enhanced by the addition of Ca. Additionally, Cd stress caused reduced uptake of essential elements and increased Cd accumulation in roots and shoots. However, application of Ca enhanced the concentration of essential elements and decreased Cd accumulation in Cd-stressed plants. Our results indicated that application of Ca enables mustard plant to withstand the deleterious effect of Cd, resulting in improved growth and seed quality of mustard plants.
PMCID: PMC4309397  PMID: 25629695
20.  Comparative Proteomics Analyses of Kobresia pygmaea Adaptation to Environment along an Elevational Gradient on the Central Tibetan Plateau 
PLoS ONE  2014;9(6):e98410.
Variations in elevation limit the growth and distribution of alpine plants because multiple environmental stresses impact plant growth, including sharp temperature shifts, strong ultraviolet radiation exposure, low oxygen content, etc. Alpine plants have developed special strategies to help survive the harsh environments of high mountains, but the internal mechanisms remain undefined. Kobresia pygmaea, the dominant species of alpine meadows, is widely distributed in the Southeastern Tibet Plateau, Tibet Autonomous Region, China. In this study, we mainly used comparative proteomics analyses to investigate the dynamic protein patterns for K. pygmaea located at four different elevations (4600, 4800, 4950 and 5100 m). A total of 58 differentially expressed proteins were successfully detected and functionally characterized. The proteins were divided into various functional categories, including material and energy metabolism, protein synthesis and degradation, redox process, defense response, photosynthesis, and protein kinase. Our study confirmed that increasing levels of antioxidant and heat shock proteins and the accumulation of primary metabolites, such as proline and abscisic acid, conferred K. pygmaea with tolerance to the alpine environment. In addition, the various methods K. pygmaea used to regulate material and energy metabolism played important roles in the development of tolerance to environmental stress. Our results also showed that the way in which K. pygmaea mediated stomatal characteristics and photosynthetic pigments constitutes an enhanced adaptation to alpine environmental stress. According to these findings, we concluded that K. pygmaea adapted to the high-elevation environment on the Tibetan Plateau by aggressively accumulating abiotic stress-related metabolites and proteins and by the various life events mediated by proteins. Based on the species'lexible physiological and biochemical processes, we surmised that environment change has only a slight impact on K. pygmaea except for possible impacts to populations on vulnerable edges of the species' range.
PMCID: PMC4041879  PMID: 24887403
21.  Enhancement of the proline and nitric oxide synthetic pathway improves fermentation ability under multiple baking-associated stress conditions in industrial baker's yeast 
During the bread-making process, industrial baker's yeast, mostly Saccharomyces cerevisiae, is exposed to baking-associated stresses, such as air-drying and freeze-thaw stress. These baking-associated stresses exert severe injury to yeast cells, mainly due to the generation of reactive oxygen species (ROS), leading to cell death and reduced fermentation ability. Thus, there is a great need for a baker's yeast strain with higher tolerance to baking-associated stresses. Recently, we revealed a novel antioxidative mechanism in a laboratory yeast strain that is involved in stress-induced nitric oxide (NO) synthesis from proline via proline oxidase Put1 and N-acetyltransferase Mpr1. We also found that expression of the proline-feedback inhibition-less sensitive mutant γ-glutamyl kinase (Pro1-I150T) and the thermostable mutant Mpr1-F65L resulted in an enhanced fermentation ability of baker's yeast in bread dough after freeze-thaw stress and air-drying stress, respectively. However, baker's yeast strains with high fermentation ability under multiple baking-associated stresses have not yet been developed.
We constructed a self-cloned diploid baker's yeast strain with enhanced proline and NO synthesis by expressing Pro1-I150T and Mpr1-F65L in the presence of functional Put1. The engineered strain increased the intracellular NO level in response to air-drying stress, and the strain was tolerant not only to oxidative stress but also to both air-drying and freeze-thaw stresses probably due to the reduced intracellular ROS level. We also showed that the resultant strain retained higher leavening activity in bread dough after air-drying and freeze-thaw stress than that of the wild-type strain. On the other hand, enhanced stress tolerance and fermentation ability did not occur in the put1-deficient strain. This result suggests that NO is synthesized in baker's yeast from proline in response to oxidative stresses that induce ROS generation and that increased NO plays an important role in baking-associated stress tolerance.
In this work, we clarified the importance of Put1- and Mpr1-mediated NO generation from proline to the baking-associated stress tolerance in industrial baker's yeast. We also demonstrated that baker's yeast that enhances the proline and NO synthetic pathway by expressing the Pro1-I150T and Mpr1-F65L variants showed improved fermentation ability under multiple baking-associated stress conditions. From a biotechnological perspective, the enhancement of proline and NO synthesis could be promising for breeding novel baker's yeast strains.
PMCID: PMC3359278  PMID: 22462683
Baker's yeast; Proline; Mpr1; Nitric oxide; Baking-associated stress tolerance
22.  A Novel RNA-Recognition-Motif Protein Is Required for Premeiotic G1/S-Phase Transition in Rice (Oryza sativa L.) 
PLoS Genetics  2011;7(1):e1001265.
The molecular mechanism for meiotic entry remains largely elusive in flowering plants. Only Arabidopsis SWI1/DYAD and maize AM1, both of which are the coiled-coil protein, are known to be required for the initiation of plant meiosis. The mechanism underlying the synchrony of male meiosis, characteristic to flowering plants, has also been unclear in the plant kingdom. In other eukaryotes, RNA-recognition-motif (RRM) proteins are known to play essential roles in germ-cell development and meiosis progression. Rice MEL2 protein discovered in this study shows partial similarity with human proline-rich RRM protein, deleted in Azoospermia-Associated Protein1 (DAZAP1), though MEL2 also possesses ankyrin repeats and a RING finger motif. Expression analyses of several cell-cycle markers revealed that, in mel2 mutant anthers, most germ cells failed to enter premeiotic S-phase and meiosis, and a part escaped from the defect and underwent meiosis with a significant delay or continued mitotic cycles. Immunofluorescent detection revealed that T7 peptide-tagged MEL2 localized at cytoplasmic perinuclear region of germ cells during premeiotic interphase in transgenic rice plants. This study is the first report of the plant RRM protein, which is required for regulating the premeiotic G1/S-phase transition of male and female germ cells and also establishing synchrony of male meiosis. This study will contribute to elucidation of similarities and diversities in reproduction system between plants and other species.
Author Summary
Meiosis is a pivotal event to produce haploid spores and gametes in all sexually reproducing species and is a fundamentally different type of cell cycle from mitosis. Thus, the molecular mechanisms to switch the cell cycle from mitosis to meiosis have been studied by many researchers. In yeast and metazoans, RNA-binding proteins are known to play important roles in the post-transcriptional regulation of genes implicated in the meiotic entry and meiosis. In contrast, in the plant kingdom, the mechanisms to control the meiotic entry have largely remained elusive. In this study, we discover a novel RNA-recognition-motif (RRM) protein in rice (Oryza sativa L.), designated MEL2, and demonstrate that MEL2 is required for the faithful transition of germ cells from mitosis to meiotic cell cycle. Rice MEL2 shows partial similarity with human DAZAP1, which is an RRM protein and relates to Azoospermia syndrome in human, while there are critical structural differences between germline-specific RRM proteins of mammals and plants. Our findings will lead the molecular-biological studies of plant meiotic entry to the next steps and will enable a comparison of the systems of meiotic entry between animals and plants.
PMCID: PMC3017114  PMID: 21253568
23.  Tobacco seeds expressing feedback-insensitive cystathionine gamma-synthase exhibit elevated content of methionine and altered primary metabolic profile 
BMC Plant Biology  2013;13:206.
The essential sulfur-containing amino acid methionine plays a vital role in plant metabolism and human nutrition. In this study, we aimed to elucidate the regulatory role of the first committed enzyme in the methionine biosynthesis pathway, cystathionine γ-synthase (CGS), on methionine accumulation in tobacco seeds. We also studied the effect of this manipulation on the seed’s metabolism.
Two forms of Arabidopsis CGS (AtCGS) were expressed under the control of the seeds-specific promoter of legumin B4: feedback-sensitive F-AtCGS (LF seeds), and feedback-insensitive T-AtCGS (LT seeds). Unexpectedly, the soluble content of methionine was reduced significantly in both sets of transgenic seeds. Amino acids analysis and feeding experiments indicated that although the level of methionine was reduced, the flux through its synthesis had increased. As a result, the level of protein-incorporated methionine had increased significantly in LT seeds by up to 60%, but this was not observed in LF seeds, whose methionine content is tightly regulated. This increase was accompanied by a higher content of other protein-incorporated amino acids, which led to 27% protein content in the seeds although this was statistically insignificantly. In addition, the levels of reducing sugars (representing starch) were slightly but significantly reduced, while that of oil was insignificantly reduced. To assess the impact of the high expression level of T-AtCGS in seeds on other primary metabolites, metabolic profiling using GC-MS was performed. This revealed significant alterations to the primary seed metabolism manifested by a significant increase in eight annotated metabolites (mostly sugars and their oxidized derivatives), while the levels of 12 other metabolites were reduced significantly in LT compared to wild-type seeds.
Expression of T-AtCGS leads to an increase in the level of total Met, higher contents of total amino acids, and significant changes in the levels of 20 annotated metabolites. The high level of oxidized metabolites, the two stress-associated amino acids, proline and serine, and low level of glutathione suggest oxidative stress that occurs during LT seed development. This study provides information on the metabolic consequence of increased CGS activity in seeds and how it affects the seed’s nutritional quality.
PMCID: PMC3878949  PMID: 24314105
Amino acids; Cystathionine γ-synthase; Germination; Methinonine; Metabolism; Seeds; Storage proteins
24.  Desensitization of Feedback Inhibition of the Saccharomyces cerevisiae γ-Glutamyl Kinase Enhances Proline Accumulation and Freezing Tolerance▿  
Applied and Environmental Microbiology  2007;73(12):4011-4019.
In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. The yeast Saccharomyces cerevisiae induces trehalose or glycerol synthesis but does not increase intracellular proline levels during various stresses. Using a proline-accumulating mutant, we previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. This mutant was recently shown to carry an allele of PRO1 which encodes the Asp154Asn mutant γ-glutamyl kinase (GK), the first enzyme of the proline biosynthetic pathway. Here, enzymatic analysis of recombinant proteins revealed that the GK activity of S. cerevisiae is subject to feedback inhibition by proline. The Asp154Asn mutant was less sensitive to feedback inhibition than wild-type GK, leading to proline accumulation. To improve the enzymatic properties of GK, PCR random mutagenesis in PRO1 was employed. The mutagenized plasmid library was introduced into an S. cerevisiae non-proline-utilizing strain, and proline-overproducing mutants were selected on minimal medium containing the toxic proline analogue azetidine-2-carboxylic acid. We successfully isolated several mutant GKs that, due to extreme desensitization to inhibition, enhanced the ability to synthesize proline better than the Asp154Asn mutant. The amino acid changes were localized at the region between positions 142 and 154, probably on the molecular surface, suggesting that this region is involved in allosteric regulation. Furthermore, we found that yeast cells expressing Ile150Thr and Asn142Asp/Ile166Val mutant GKs were more tolerant to freezing stress than cells expressing the Asp154Asn mutant.
PMCID: PMC1932739  PMID: 17449694
25.  Cell-type specific analysis of translating RNAs in developing flowers reveals new levels of control 
Combining translating ribosome affinity purification with RNA-seq for cell-specific profiling of translating RNAs in developing flowers.Cell type comparisons of cell type-specific hormone responses, promoter motifs, coexpressed cognate binding factor candidates, and splicing isoforms.Widespread post-transcriptional regulation at both the intron splicing and translational stages.A new class of noncoding RNAs associated with polysomes.
What constitutes a differentiated cell type? How much do cell types differ in their transcription of genes? The development and functions of tissues rely on constant interactions among distinct and nonequivalent cell types. Answering these questions will require quantitative information on transcriptomes, proteomes, protein–protein interactions, protein–nucleic acid interactions, and metabolomes at cellular resolution. The systems approaches emerging in biology promise to explain properties of biological systems based on genome-wide measurements of expression, interaction, regulation, and metabolism. To facilitate a systems approach, it is essential first to capture such components in a global manner, ideally at cellular resolution.
Recently, microarray analysis of transcriptomes has been extended to a cellular level of resolution by using laser microdissection or fluorescence-activated sorting (for review, see Nelson et al, 2008). These methods have been limited by stresses associated with cellular separation and isolation procedures, and biases associated with mandatory RNA amplification steps. A newly developed method, translating ribosome affinity purification (TRAP; Zanetti et al, 2005; Heiman et al, 2008; Mustroph et al, 2009), circumvents these problems by epitopetagging a ribosomal protein in specific cellular domains to selectively purify polysomes. We combined TRAP with deep sequencing, which we term TRAP-seq, to provide cell-level spatiotemporal maps for Arabidopsis early floral development at single-base resolution.
Flower development in Arabidopsis has been studied extensively and is one of the best understood aspects of plant development (for review, see Krizek and Fletcher, 2005). Genetic analysis of homeotic mutants established the ABC model, in which three classes of regulatory genes, A, B and C, work in a combinatorial manner to confer organ identities of four whorls (Coen and Meyerowitz, 1991). Each class of regulatory gene is expressed in a specific and evolutionarily conserved domain, and the action of the class A, B and C genes is necessary for specification of organ identity (Figure 1A).
Using TRAP-seq, we purified cell-specific translating mRNA populations, which we and others call the translatome, from the A, B and C domains of early developing flowers, in which floral patterning and the specification of floral organs is established. To achieve temporal specificity, we used a floral induction system to facilitate collection of early stage flowers (Wellmer et al, 2006). The combination of TRAP-seq with domain-specific promoters and this floral induction system enabled fine spatiotemporal isolation of translating mRNA in specific cellular domains, and at specific developmental stages.
Multiple lines of evidence confirmed the specificity of this approach, including detecting the expression in expected domains but not in other domains for well-studied flower marker genes and known physiological functions (Figures 1B–D and 2A–C). Furthermore, we provide numerous examples from flower development in which a spatiotemporal map of rigorously comparable cell-specific translatomes makes possible new views of the properties of cell domains not evident in data obtained from whole organs or tissues, including patterns of transcription and cis-regulation, new physiological differences among cell domains and between flower stages, putative hormone-active centers, and splicing events specific for flower domains (Figure 2A–D). Such findings may provide new targets for reverse genetics studies and may aid in the formulation and validation of interaction and pathway networks.
Beside cellular heterogeneity, the transcriptome is regulated at several steps through the life of mRNA molecules, which are not directly available through traditional transcriptome profiling of total mRNA abundance. By comparing the translatome and transcriptome, we integratively profiled two key posttranscriptional control points, intron splicing and translation state. From our translatome-wide profiling, we (i) confirmed that both posttranscriptional regulation control points were used by a large portion of the transcriptome; (ii) identified a number of cis-acting features within the coding or noncoding sequences that correlate with splicing or translation state; and (iii) revealed correlation between each regulation mechanism and gene function. Our transcriptome-wide surveys have highlighted target genes transcripts of which are probably under extensive posttranscriptional regulation during flower development.
Finally, we reported the finding of a large number of polysome-associated ncRNAs. About one-third of all annotated ncRNA in the Arabidopsis genome were observed co-purified with polysomes. Coding capacity analysis confirmed that most of them are real ncRNA without conserved ORFs. The group of polysome-associated ncRNA reported in this study is a potential new addition to the expanding riboregulator catalog; they could have roles in translational regulation during early flower development.
Determining both the expression levels of mRNA and the regulation of its translation is important in understanding specialized cell functions. In this study, we describe both the expression profiles of cells within spatiotemporal domains of the Arabidopsis thaliana flower and the post-transcriptional regulation of these mRNAs, at nucleotide resolution. We express a tagged ribosomal protein under the promoters of three master regulators of flower development. By precipitating tagged polysomes, we isolated cell type-specific mRNAs that are probably translating, and quantified those mRNAs through deep sequencing. Cell type comparisons identified known cell-specific transcripts and uncovered many new ones, from which we inferred cell type-specific hormone responses, promoter motifs and coexpressed cognate binding factor candidates, and splicing isoforms. By comparing translating mRNAs with steady-state overall transcripts, we found evidence for widespread post-transcriptional regulation at both the intron splicing and translational stages. Sequence analyses identified structural features associated with each step. Finally, we identified a new class of noncoding RNAs associated with polysomes. Findings from our profiling lead to new hypotheses in the understanding of flower development.
PMCID: PMC2990639  PMID: 20924354
Arabidopsis; flower; intron; transcriptome; translation

Results 1-25 (1015522)