Ghrelin in plants: what is the function of an appetite hormone in plants?

In the present work, we provide compelling evidence for the expression of a ghrelin-like peptide hormone that has only been associated with animals, in various plant tissues. Ghrelin, the appetite stimulating hormone, has been identified from a number of different species including humans, rat, pig, mouse, gerbil, eel, goldfish, bullfrog and chicken. The study here was conducted using an immunohistochemistry assay to screen whether plants have any ghrelin immunoreactivity. In this respect, Prunus x domestica L. and Marus alba were examined. Immunohistochemistry results showed that there is a strong human ghrelin immunoreactivity substance in the parenchyma cells of these plants. This was entirely unexpected since this hormone was considered to be present solely in animals. Thus, this study is the first to report the presence of a peptide with ghrelin-like activity in plants, a finding that has only been observed in the animal kingdom. RIA analysis confirmed that these plants contain significant amounts of this substance. Furthermore, reverse-phase HPLC analyses of plant extracts showed an elution characteristic of the peptide identical to that of human ghrelin. In general, fruit from both plants had higher levels of the peptide than the vegetative parts.     

Rhomboid proteases in plants - still in square one?

Rhomboids are ubiquitous intramembrane serine proteases the sequences of which are found in nearly all sequenced genomes, including those of plants. They were molecularly characterized in a number of organisms, and were found to play a role in a variety of biological functions including signaling, development, apoptosis, mitochondrial integrity, parasite invasion and more. Although rhomboid sequences are found in plants, very little is known about their function. Here, we present the current knowledge in the rhomboids field in general, and in plant rhomboids in particular. In addition, we discuss possible physiological roles of different plant rhomboids.     

Sexual devolution in plants: apomixis uncloaked?

There are a growing number of examples where naturally occurring mutations disrupt an established physiological or developmental pathway to yield a new condition that is evolutionary favored. Asexual reproduction by seed in plants, or apomixis, occurs in a diversity of taxa and has evolved from sexual ancestors. One form of apomixis, diplospory, is a multi-step development process that is initiated when meiosis is altered to produce an unreduced rather than a reduced egg cell. Subsequent parthenogenetic development of the unreduced egg yields genetically maternal progeny. While it has long been apparent from cytological data that meiosis in apomicts was malfunctional or completely bypassed, the genetic basis of the phenomenon has been a long-standing mystery. New data from genetic analysis of Arabidopsis mutants in combination with more sophisticated molecular understanding of meiosis in plants indicate that a weak mutation of the gene SWI, called DYAD, interferes with sister chromatid cohesion in meiosis I, causes synapsis to fail in female meiosis and yields two unreduced cells. The new work shows that a low percentage of DYAD ovules produce functional unreduced egg cells (2n) that can be fertilized by haploid pollen (1n) to give rise to triploid (3n) progeny. While the DYAD mutants differ in some aspects from naturally occurring apomicts, the work establishes that mutation to a single gene can effectively initiate apomictic development and, furthermore, focuses efforts to isolate apomixis genes on a narrowed set of developmental events. Profitable manipulation of meiosis and recombination in agronomically important crops may be on the horizon.     

Natural products – modifying metabolite pathways in plants

The diversity of plant natural product (PNP) molecular structures is reflected in the variety of biochemical and genetic pathways that lead to their formation and accumulation. Plant secondary metabolites are important commodities, and include fragrances, colorants, and medicines. Increasing the extractable amount of PNP through plant breeding, or more recently by means of metabolic engineering, is a priority. The prerequisite for any attempt at metabolic engineering is a detailed knowledge of the underlying biosynthetic and regulatory pathways in plants. Over the past few decades, an enormous body of information about the biochemistry and genetics of biosynthetic pathways involved in PNPs production has been generated. In this review, we focus on the three large classes of plant secondary metabolites: terpenoids (or isoprenoids), phenylpropanoids, and alkaloids. All three provide excellent examples of the tremendous efforts undertaken to boost our understanding of biosynthetic pathways, resulting in the first successes in plant metabolic engineering. We further consider what essential information is still missing, and how future research directions could help achieve the rational design of plants as chemical factories for high-value products.     

Müllerian mimicry in aposematic spiny plants

Müllerian mimicry is common in aposematic animals but till recently, like other aspects of plant aposematism was almost unknown. Many thorny, spiny and prickly plants are considered aposematic because their sharp defensive structures are colorful and conspicuous. Many of these spiny plant species (e.g., cacti and Agave in North American deserts; Aloe, Euphorbia and acacias with white thorns in Africa; spiny plants in Ohio; and spiny members of the Asteraceae in the Mediterranean basin) have overlapping territories, and also similar patterns of conspicuous coloration, and suffer from the evolutionary pressure of grazing by the same large herbivores. I propose that many of these species form Müllerian mimicry rings.Key words: aposematic coloration, defense, evolution, herbivory, müllerian mimicry, spines, thornsAposematic (warning) coloration is a biological phenomenon in which poisonous, dangerous or otherwise unpalatable organisms visually advertise these qualities to other animals. The evolution of aposematic coloration is based on the ability of target enemies to associate the visual signal with the risk, damage or non-profitable handling, and later to avoid such organisms as prey. Typical colors of aposematic animals are yellow, orange, red, purple, black, white or brown and combinations of these.^1–5 Many thorny, spiny and prickly plant species were proposed to be aposematic because their sharp defensive structures are usually colorful (yellow, orange, red, brown, black, white) and/or associated with similar conspicuous coloration.^5–22 Animal spines also have similar conspicuous coloration and were proposed to be aposematic.^1,5,17,23Several authors have proposed that mimicry of various types helps in plant defense, e.g.,^9,24–34 More specifically, Müllerian mimicry was already proposed to exist in several defensive plant signaling systems. The first was for several spiny species with white-variegated leaves.^8,10 The second was for some tree species with red or yellow poisonous autumn leaves.³⁵ The third cases are of a mixture of Müllerian and Batesian mimicry, of thorn auto-mimicry found in many Agave species.⁸Here I propose that many species of visually aposematic spiny plants of the following taxa: (1) Cactaceae, (2) the genus Agave, (3) the genus Aloe, (4) African thorny members of the genus Euphorbia, (5) African acacias with white thorns, (6) spiny vascular plants of southeastern Ohio, (7) spiny Near Eastern plants with white variegation on their leaves, (8) Near Eastern members of the Asteraceae with yellow spines, form Müllerian mimicry rings of spiny plants.To consider the existence of Müllerian mimicry rings in aposematic organisms, two factors are needed: (1) a similar signal, and (2) an overlapping distribution in respect to the territory of predators in animals, or herbivores in plants. I will show below that for the plant taxa proposed here to form Müllerian mimicry rings, both criteria operate.The accumulating data about the common association of plant defenses by spines with visual conspicuousness, along with the fact that many such species overlap in their habitat, raises the possibility of the broad phenomenon of existence of Müllerian mimicry rings in plants. Even from the limited number of publications proposing visual aposematism in spiny plants, the operation of vegetal Müllerian mimicry rings seems to be obvious. The phenomenon can now be traced to both the Old World (Asia, Africa and Europe) and the New World (North America). The best-studied cases include Cactaceae and the genera Agave, Aloe and Euphorbia,⁶ African acacias with white thorns,^12,15 Near Eastern spiny plants with white variegation on their leaves,^7,11 aposematic spiny vascular plants of southeastern Ohio,¹⁶ and many spiny Mediterranean species of the Asteraceae with yellow spines.²²In the four spiny taxa (Cactaceae and the genera Agave, Aloe and Euphorbia) that were the first to be proposed as visually aposematic⁶ there is a very strong morphological similarity. In cacti, there are two types of conspicuousness of spines that are typical of many plant species: (1) colorful spines, and (2) white spots, or white or colorful stripes, associated with spines on the stems. These two types of aposematic coloration also dominate the spine system of Agave, Aloe and Euphorbia. The fact that many species of three of these four spiny taxa (Agave, Aloe and Euphorbia) are also poisonous^36–38 further indicates their potential to form Müllerian mimicry rings.I propose that each of these groups for itself and some of these groups (e.g., Cactaceae and the genus Agave in North America; Aloe, Euphorbia and acacias in east and south Africa) that have overlapping distribution and share at least some of the herbivores, form Müllerian mimicry rings.The first Müllerian mimicry ring is of cacti and Agave that have an overlapping distribution over large areas in North America.^37,39 The large herbivores in North America disappeared not so long ago in evolutionary time scales and seem to have shaped the spiny defense of these plant taxa.⁴⁰The second Müllerian mimicry ring is of the spiny and thorny members of the African genera Aloe, Euphorbia and certain acacias with very conspicuous white thorns, which partly overlap in distribution and share various large mammalian herbivores.^12,15,36,41The third Müllerian mimicry ring is the outcome of the common presence of aposematic coloration in spiny vascular plants of southeastern Ohio,¹⁶ with color patterns in thorns and spines similar to those of Cactaceae and the genera Agave, Aloe and Euphorbia described in Lev-Yadun.⁶The next case of potential operation of Müllerian mimicry ring of spiny plants with overlapping territories that suffer from the same large herbivores, but on a much smaller geographical scale, has recently been proposed for several spiny species with white-variegated leaves,⁷ and later for more than 20 spiny species in the flora of Israel that have white markings associated with their spines.¹¹The last case of a probable Müllerian mimicry ring was described by Ronel et al.²² who while studying the spine system of Near Eastern spiny members of the Asteraceae, found 29 spiny species with yellow spines, and additional such species are expected to occur. Since some of these species and others with yellow spines also grow in southern Europe, it is clear that the same phenomenon is also common there.I conclude that Müllerian mimicry rings seem to be very common in plants, and that it is probable that many other spiny plants that form Müllerian mimicry rings are waiting to be studied. Such defensive rings are probably also formed by poisonous plants that share similar colors or odors.     

Are there multiple circadian clocks in plants?

We have reported that Arabidopsis might have genetically distinct circadian oscillators in multiple cell-types.¹ Rhythms of CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are 2.5 h longer in phytochromeB mutants in constant red light and in cryptocrome1 cry2 double mutant (hy4-1 fha-1) in constant blue light than the wild-type.² However, we found that cytosolic free Ca²⁺ ([Ca²⁺]_cyt) oscillations were undetectable in these mutants in the same light conditions.¹ Furthermore, mutants of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) have short period rhythms of leaf movement but have arrhythmic [Ca²⁺]_cyt oscillations. More important, the timing of cab1-1 (toc1-1) mutant has short period rhythms of CAB2 promoter activity (∼21 h) but, surprisingly, has a wild-type period for circadian [Ca²⁺]_cyt oscillations (∼24 h). In contrast, toc1-2, a TOC1 loss-of-function mutant, has a short period of both CAB2 and [Ca²⁺]_cyt rhythms (∼21 h). Here we discuss the difference between the phenotypes of toc1-1 and toc1-2 and how rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations might be regulated differently.Key words: circadian rhythms, TOC1, multiple oscillators, CAB2, Ca²⁺ signalling, arabidopsis, circadian [Ca²⁺]_cyt oscillations, aequorin, luciferase, central oscillatorThe plant circadian clock controls a multitude of physiological processes such as photosynthesis, organ and stomatal movements and transition to reproductive growth. A plant clock that is correctly matched to the rhythms in the environment brings about a photosynthetic advantage that results in more chlorophyll, more carbon assimilation and faster growth.³ One of the first circadian clock mutants to be described in plants was the short period timing of cab1-1 (toc1-1), which was identified using the rhythms of luciferase under a CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter as a marker for circadian period.⁴Circadian rhythms of both CAB2 promoter activity and cytosolic-free Ca²⁺ ([Ca²⁺]_cyt) oscillations depend on the function of a TOC1, CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL (TOC1/CCA1/LHY) negative feedback loop.⁵ In tobacco seedlings, CAB2:luciferase (CAB2:luc) rhythms and circadian [Ca²⁺]_cyt oscillations can be uncoupled in undifferentiated calli.⁶ In Arabidopsis, we reported that toc1-1 has different periods of rhythms of CAB2 promoter activity (∼21 h) and circadian [Ca²⁺]_cyt oscillations (∼24 h). The mutant allele toc1-1 has a base pair change that leads to a full protein that has an amino acid change from Ala to Val in the CCT domain (CONSTANS, CONSTANS-LIKE and TOC1).⁷ On the other hand, the mutant toc1-2 has short period of both rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations (∼21 h).^1,7 This allele has a base pair change that results in changes to preferential mRNA splicing, resulting in a truncated protein with only 59 residues.⁷ Thus, the mutated CCT domain in toc1-1 might lead to the uncoupling of rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations while the absence of TOC1 in toc1-2 causes the shortening of the period of both rhythms. Indeed, zeitlupe-1 (ztl-1) mutants, that have higher levels of TOC1, have long periods of both rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations.¹ The biochemical function of the CCT domain is unknown but it is predicted to play an important role in protein-protein interactions⁸ and nuclear localization.⁹One model to explain the period difference of CAB2:luc expression and circadian [Ca²⁺]_cyt oscillation is that the toc1-1 mutation has uncoupled two oscillators in the same cell. Uncoupled oscillators are a predicted outcome of certain mutations in the recently described three-loop mathematical model.^10–11 However, both rhythms of TOC1 and CCA1/LHY expression, which would be in uncoupled oscillators accordingly to the model, are described as short-period in toc1-1.⁵ Thus, we have favored the model in which CAB2:luc expression and circadian [Ca²⁺]_cyt oscillation are reporting cell-types with different oscillators that are affected differently by toc1-1.It is possible that TOC1 could interact with a family of cell-type specific proteins. The interaction of TOC1 with each member of the family could be affected differently by the mutation in the CCT domain (Fig. 1). Two-hybrid assays have shown that TOC1 interacts with PIF proteins (PHYTOCHROME INTERACTING FACTOR3 and PIF4) and related PIL proteins (PIF3-LIKE PROTEIN 1, PIL2, PIL5 and PIL6).⁸ In fact, TOC1 interaction with both PIF3 and PIL1 is stronger when the N-terminus receiver domain is taken out and the CCT domain is left intact.⁸ Thus, it is possible that TOC1 and different PIF/PIL proteins interact to regulate the central oscillator. This interaction could be impaired by the Ala to Val change in the toc1-1 mutation, leading to the period shortening. However, lines misexpressing PIF3, PIL1 and PIL6 showed no changes in their circadian rhythms.^12–16Open in a separate windowFigure 1Models of how the toc1-1 mutation might differently affect cell-type specific circadian oscillators. The single mutant toc1-1 have 21 h rhythms of CAB2 promoter activity and 24 h-rhythms of [Ca²⁺]_cyt oscillations. The toc1-1 mutation is a single amino acid change in the CCT domain. The CCT domain is involved in protein-protein interaction and/or nuclear localization. We have proposed that circadian oscillators with different periods are present in different cell-types. The luminescence generated by CAB2 promoter-drived luciferase (from the CAB2:luc) is probably originated in the epidermis and mesophyll cells. In this model, we propose that the mutation on the CCT domain impairs the mutated TOC1 interaction with the hypothetical protein Z in these cells-types. In contrast, in other cell-types, the mutated TOC1 still interacts with other hypothetical proteins (W), despite the mutation in the CCT domain. In those cell-types, the circadian oscillator could still run with a 24 h period for [Ca²⁺]_cyt rhythms (from the 35S:AEQ construct). One possible identity for Z and W are the members of the PHYTOCHROME INTERACTING FACTOR (PIF) related PIF3-LIKE (PIL) family.One possible explanation for the absence of alterations in the period of circadian rhythms in lines misexpressing PIF/PIL is that they only have roles in certain cell-types. As an example, PIL6 and PIF3 are involved with flowering time and hypocotyl growth in red light^12–15 while PIL1 and PIL2 are involved with hypocotyl elongation in shade-avoidance responses.¹⁶ Both hypocotyl growth and flowering time require cell-type specific regulation: vascular bundle cells in the case of the flowering time¹⁷ and the cells in the shoot in the case of the hypocotyl elongation.¹⁶ If TOC1 interaction with certain PIF/PIL is indeed cell-type specific, the mutated CCT domain found in the toc1-1 mutant could affect the clock in different ways, depending on the type of PIF/PIL protein expressed in each cell-type. Therefore, a question that arises is: which cell-types are sensitive to the toc1-1 mutation?There is evidence that CAB2 and CATALASE3 (CAT3) are regulated by two oscillators that respond differently to temperature signals.¹⁸ These genes might be regulated by two distinct circadian oscillators within the same tissues or a single cell.¹⁸ Interestingly, the spatial patterns of expression of CAB2 and CATALASE3 overlap in the mesophyll of the cotyledons.¹⁸ Furthermore, rhythms of CAB2 and CHALCONE SYNTHASE (CHS) promoter activity have different periods and they are equally affected by toc1-1 mutation.¹⁹ Whereas CAB2 is mainly expressed in the mesophyll cells, CHS is mainly expressed in epidermis and root cells.¹⁹ However, rhythms of AEQUORIN luminescence, which reports [Ca²⁺]_cyt oscillation, were insensitive to toc1-1 mutation and appear to come from the whole cotyledon.²⁰ One cell-type which is found in the whole cotyledon but is distinct from either mesophyll or epidermis cells is the vascular tissue and associated cells.Another approach to determine which cell-types are insensitive to toc1-1 mutation is to compare the toc1-1 and toc1-2 phenotypes. The period of circadian [Ca²⁺]_cyt oscillations is not the only phenotype that is different in toc1-1 and toc1-2 mutants. Rhythms in CAB2 promoter activity in constant red light are short period in toc1-1 but arrhythmic in toc1-2.^21,22 COLD, CIRCADIAN RHYTHM AND RNA BINDING 2/GLYCINE-RICH RNA BINDING PROTEIN 7 (CCR2/GRP7) is also arrhythmic in toc1-2 but short period in toc1-1 in constant darkness.^7,22 When the length of the hypocotyl was measured for both toc1-1 and toc1-2 plants exposed to various intensities of red light, only toc1-2 had a clear reduction in sensitivity to red light. Therefore, toc1-2 has long hypocotyl when maintained in constant red light while hypocotyl length in toc1-1 is nearly identical to that in the wild-type.²² These differences may allow us to separate which cell-types are sensitive to the toc1-1 mutation and which not.Hypocotyl growth is regulated by a large number of factors such as light, gravity, auxin, cytokinins, ethylene, gibberellins and brassinosteroids.²³ There is also a correlation between the size of the hypocotyl in red light and defects in the circadian signaling network.^24,25 The fact that toc1-1 has different hypocotyl sizes from toc1-2 suggests that circadian [Ca²⁺]_cyt oscillations could be involved in the light-dependent control of hypocotyl growth. Circadian [Ca²⁺]_cyt oscillations might encode temporal information to control cell expansion and hypocotyl growth.^26–28 toc1-1 have short-period rhythms of hypocotyl elongation, which indicates that the cells in the hypocotyl have a 21 h oscillator.²⁹ However, toc1-1 might also have a wild-type hypocotyl length in continuous red light because cells which generate the signal to regulate hypocotyl growth might have 24 h oscillators.The toc1-1 mutation was the first to be directly associated with the plant circadian clock, revitalizing the field of study.⁴ Now, by either uncoupling two feedback loops or by distinct TOC1 protein-protein interaction in different cell-types, toc1-1 has shown new properties of the circadian clock that may deepen our understanding of this system.     

Can mechanics control pattern formation in plants?

Development of the plant body entails many pattern forming events at scales ranging from the cellular level to the whole plant. Recent evidence suggests that mechanical forces play a role in establishing some of these patterns. The development of cellular configurations in glandular trichomes and the rippling of leaf surfaces are discussed in depth to illustrate how intricate patterns can emerge from simple and well-established molecular and cellular processes. The ability of plants to sense and transduce mechanical signals suggests that complex interactions between mechanics and chemistry are possible during plant development. The inclusion of mechanics alongside traditional molecular controls offers a more comprehensive view of developmental processes.     

Is there leaderless protein secretion in plants?

The sugar alcohol mannitol and it’s catabolic enzyme mannitol dehydrogenase (MTD), in addition to welldocumented roles in metabolism and osmoprotection, may play roles in hostpathogen interactions. Research suggests that in response to the mannitol that pathogenic fungi secrete to suppress reactive oxygen-mediated host defenses, plants make MTD to catabolize fungal mannitol. Yet previous work suggested that pathogen-secreted mannitol is extracellular, while in healthy plants MTD is cytoplasmic. We have presented results showing that the normally cytoplasmic MTD is exported into the cell wall or extracellular space in response to the endogenous inducer of plant defense responses salicylic acid (SA). This SA-induced secretion is insensitive to brefeldin A, an inhibitor of Golgimediated protein transport. Together with the absence of MTD in Golgi stacks and the lack of a documented extracellular targeting sequence in the MTD protein, this suggests MTD is secreted by a non-Golgi, pathogen-activated secretion mechanism in plants. Here we discuss the potential significance of non-Golgi secretion in response to stress.Key words: protein secretion, mannitol metabolism, plant-pathogen interaction, extracellular space, apoplast     

How to characterize meiotic functions in plants?

Our understanding of plant meiosis is rapidly increasing thanks to the model Arabidopsis thaliana. Here we present the results of a screening for meiotic mutants carried out with a library containing 30,719 T-DNA insertion lines. An average of one mutant per 1000 lines was recovered. Several phenotypic classes could be distinguished and are presented. In parallel, 39 proteins known to be involved in meiosis in non-plant organisms were chosen and a search was performed for homologue sequences in the completed Arabidopsis thaliana genome. Approximately 30% of the meiotic related sequences showed similarities with one or several Arabidopsis putative genes. The relevance of forward versus reverse genetics in order to characterize meiotic functions is discussed.     

Allometry of sexual size dimorphism in dioecious plants: do plants obey Rensch's rule?

Rensch's rule refers to a pattern in sexual size dimorphism (SSD) in which SSD decreases with body size when females are the larger sex and increases with body size when males are the larger sex. Many animal taxa conform to Rensch's rule, but it has yet to be investigated in plants. Using herbarium collections from New Zealand, we characterized the size of leaves and stems of 297 individuals from 38 dioecious plant species belonging to three distantly related phylogenetic lineages. Statistical comparisons of leaf sizes between males and females showed evidence for Rensch's rule in two of the three lineages, indicating SSD decreases with leaf size when females produce larger leaves and increases with leaf size when males produce larger leaves. A similar pattern in SSD was observed for stem sizes. However, in this instance, females of small-stemmed species produced much larger stems than did males, but as stem sizes increased, SSD often disappeared. We hypothesize that sexual dimorphism in stem sizes results from selection for larger stems in females, which must provide mechanical support for seeds, fruits, and dispersal vectors, and that scaling relationships in leaf sizes result from correlated evolution with stem sizes. The overall results suggest that selection for larger female stem sizes to support the weight of offspring can give rise to Rensch's rule in dioecious plants.