Liverwort oil bodies: diversity, biochemistry, and molecular cell biology of the earliest secretory structure of land plants.

Liverworts are known for their large chemical diversity. Much of this diversity is synthesized and enclosed within oil bodies (OBs), a synapomorphy of the lineage. OBs contain the enzymes to biosynthesize and store large quantities of sesquiterpenoids and other compounds while limiting their cytotoxicity. Recent important biochemical and molecular discoveries related to OB formation, diversity, and biochemistry allow comparison with other secretory structures of land plants from an evo-devo perspective. This review addresses and discusses the most recent advances in OB origin, development, and function towards understanding the importance of these organelles in liverwort physiology and adaptation to changing environments. Our mapping of OB types and chemical compounds to the current liverwort phylogeny suggests that OBs were present in the most recent common ancestor of liverworts, supporting that OBs evolved as the first secretory structures in land plants. Yet, we require better sampling to define the macroevolutionary pattern governing the ancestral type of OB. We conclude that current efforts to find molecular mechanisms responsible for the morphological and chemical diversity of secretory structures will help understand the evolution of each major group of land plants, and open new avenues in biochemical research on bioactive compounds in bryophytes and vascular plants.

ROS-Scavenging Enzymes as an Antioxidant Response to High Concentration of Anthracene in the Liverwort Marchantia polymorpha L.

Marchantia polymorpha L. responds to environmental changes using a myriad set of physiological responses, some unique to the lineage related to the lack of a vascular- and root-system. This study investigates the physiological response of M. polymorpha to high doses of anthracene analysing the antioxidant enzymes and their relationship with the photosynthetic processes, as well as their transcriptomic response. We found an anthracene dose-dependent response reducing plant biomass and associated to an alteration of the ultrastructure of a 23.6% of chloroplasts. Despite a reduction in total thallus-chlorophyll of 31.6% of Chl a and 38.4% of Chl b, this was not accompanied by a significant change in the net photosynthesis rate and maximum quantum efficiency (Fv/Fm). However, we found an increase in the activity of main ROS-detoxifying enzymes of 34.09% of peroxidase and 692% of ascorbate peroxidase, supported at transcriptional level with the upregulation of ROS-related detoxifying responses. Finally, we found that M. polymorpha tolerated anthracene-stress under the lowest concentration used and can suffer physiological alterations under higher concentrations tested related to the accumulation of anthracene within plant tissues. Our results show that M. polymorpha under PAH stress condition activated two complementary physiological responses including the activation of antioxidant mechanisms and the accumulation of the pollutant within plant tissues to mitigate the damage to the photosynthetic apparatus.

Molecular mechanisms involved in functional macroevolution of plant transcription factors.

Transcription factors (TFs) are key components of the transcriptional regulation machinery. In plants, they accompanied the evolution from unicellular aquatic algae to complex flowering plants that dominate the land environment. The adaptations of the body plan and physiological responses required changes in the biological functions of TFs. Some ancestral gene regulatory networks are highly conserved, while others evolved more recently and only exist in particular lineages. The recent emergence of novel model organisms provided the opportunity for comparative studies, producing new insights to infer these evolutionary trajectories. In this review, we comprehensively revisit the recent literature on TFs of nonseed plants and algae, focusing on the molecular mechanisms driving their functional evolution. We discuss the particular contribution of changes in DNA-binding specificity, protein-protein interactions and cis-regulatory elements to gene regulatory networks. Current advances have shown that these evolutionary processes were shaped by changes in TF expression pattern, not through great innovation in TF protein sequences. We propose that the role of TFs associated with environmental and developmental regulation was unevenly conserved during land plant evolution.

The Intrinsically Disordered Protein CARP9 Bridges HYL1 to AGO1 in the Nucleus to Promote MicroRNA Activity.

In plants, small RNAs are loaded into ARGONAUTE (AGO) proteins to fulfill their regulatory functions. MicroRNAs (miRNAs), one of the most abundant classes of endogenous small RNAs, are preferentially loaded into AGO1. Such loading, long believed to happen exclusively in the cytoplasm, was recently proposed to also occur in the nucleus. Here, we identified CONSTITUTIVE ALTERATIONS IN THE SMALL RNAS PATHWAYS9 (CARP9), a nuclear-localized, intrinsically disordered protein, as a factor promoting miRNA activity in Arabidopsis (Arabidopsis thaliana). Mutations in the CARP9-encoding gene led to a mild reduction of miRNAs levels, impaired gene silencing, and characteristic morphological defects, including young leaf serration and altered flowering time. Intriguingly, we found that CARP9 was able to interact with HYPONASTIC LEAVES1 (HYL1), but not with other proteins of the miRNA biogenesis machinery. In the same way, CARP9 appeared to interact with mature miRNA, but not with primary miRNA, positioning it after miRNA processing in the miRNA pathway. CARP9 was also able to interact with AGO1, promoting its interaction with HYL1 to facilitate miRNA loading in AGO1. Plants deficient in CARP9 displayed reduced levels of AGO1-loaded miRNAs, partial retention of miRNA in the nucleus, and reduced levels of AGO1. Collectively, our data suggest that CARP9 might modulate HYL1-AGO1 cross talk, acting as a scaffold for the formation of a nuclear post-primary miRNA-processing complex that includes at least HYL1, AGO1, and HEAT SHOCK PROTEIN 90. In such a complex, CARP9 stabilizes AGO1 and mature miRNAs, allowing the proper loading of miRNAs in the effector complex.

Oil Body Formation in Marchantia polymorpha Is Controlled by MpC1HDZ and Serves as a Defense against Arthropod Herbivores.

The origin of a terrestrial flora in the Ordovician required adaptation to novel biotic and abiotic stressors. Oil bodies, a synapomorphy of liverworts, accumulate secondary metabolites, but their function and development are poorly understood. Oil bodies of Marchantia polymorpha develop within specialized cells as one single large organelle. Here, we show that a class I homeodomain leucine-zipper (C1HDZ) transcription factor controls the differentiation of oil body cells in two different ecotypes of the liverwort M. polymorpha, a model genetic system for early divergent land plants. In flowering plants, these transcription factors primarily modulate responses to abiotic stress, including drought. However, loss-of-function alleles of the single ortholog gene, MpC1HDZ, in M. polymorpha did not exhibit phenotypes associated with abiotic stress. Rather, Mpc1hdz mutant plants were more susceptible to herbivory, and total plant extracts of the mutant exhibited reduced antibacterial activity. Transcriptomic analysis of the mutant revealed a reduction in expression of genes related to secondary metabolism that was accompanied by a specific depletion of oil body terpenoid compounds. Through time-lapse imaging, we observed that MpC1HDZ expression maxima precede oil body formation, indicating that MpC1HDZ mediates differentiation of oil body cells. Our results indicate that M. polymorpha oil bodies, and MpC1HDZ, are critical for defense against herbivory, but not for abiotic stress tolerance. Thus, C1HDZ genes were co-opted to regulate separate responses to biotic and abiotic stressors in two distinct land plant lineages.

Evolutionary history of HOMEODOMAIN LEUCINE ZIPPER transcription factors during plant transition to land.

Plant transition to land required several regulatory adaptations. The mechanisms behind these changes remain unknown. Since the evolution of transcription factors (TFs) families accompanied this transition, we studied the HOMEODOMAIN LEUCINE ZIPPER (HDZ) TF family known to control key developmental and environmental responses. We performed a phylogenetic and bioinformatics analysis of HDZ genes using transcriptomic and genomic datasets from a wide range of Viridiplantae species. We found evidence for the existence of HDZ genes in chlorophytes and early-divergent charophytes identifying several HDZ members belonging to the four known classes (I-IV). Furthermore, we inferred a progressive incorporation of auxiliary motifs. Interestingly, most of the structural features were already present in ancient lineages. Our phylogenetic analysis inferred that the origin of classes I, III, and IV is monophyletic in land plants in respect to charophytes. However, class IIHDZ genes have two conserved lineages in charophytes and mosses that differ in the CPSCE motif. Our results indicate that the HDZ family was already present in green algae. Later, the HDZ family expanded accompanying critical plant traits. Once on land, the HDZ family experienced multiple duplication events that promoted fundamental neo- and subfunctionalizations for terrestrial life.

The antagonistic basic helix-loop-helix partners BEE and IBH1 contribute to control plant tolerance to abiotic stress.

The bHLH family is composed by canonical and non-canonical transcription factors (TFs) that differ in the presence or absence of their DNA-binding domain, respectively. Since both types of bHLH proteins are able to dimerize, their relative abundance impacts their biological activity. Among this TF family BEE and IBH are canonical and non-canonical bHLHs, respectively and previous reports indicated that BEE2 and IBH1 dimerize. Wondering whether BEE TFs participate in the abiotic stress response and how the dimerization with IBH1 could regulate their role in Arabidopsis, double bee1/bee2 and triple bee1/bee2/bee3 mutants were tested under salinity and drought stresses. The bee1/bee2/bee3 mutant showed an enhanced tolerance whereas the double mutant behaved similar to wild type plants. These results indicated that BEE genes play a role in the stress response and also put in evidence the redundancy within the BEE family. Moreover, ectopic expression of IBH1 on different mutant backgrounds improved plant tolerance to abiotic stress, independently of the background. However, the yield of these transgenic plants was penalized with abortive seeds. Our results suggest that BEE genes are negative regulators of physiological responses to abiotic stress whereas IBH1 is a positive modulator via different pathways, one of them involving BEE TFs.

Arabidopsis thaliana homeodomain-leucine zipper type I transcription factors contribute to control leaf venation patterning.

Venation patterning is a taxonomic attribute for classification of plants and it also plays a role in the interaction of plants with the environment. Despite its importance, the molecular physiology controlling this aspect of plant development is still poorly understood. Auxin plays a central role modulating the final vein network and patterning. This addendum discusses recent findings on the role of homeodomain-leucine zipper (HD-Zip) transcription factors on the regulation of leaf venation patterning. Moreno-Piovano et al. reported that ectopic expression of a sunflower HD-Zip I gene, HaHB4, increased the asymmetry of leaf venation. Even more, this work showed that auxin transport in the leaf through LAX carriers controls venation patterning. Here, we provide evidence indicating that some Arabidopsis thaliana HD-Zip I genes play a role in the determination of the final leaf venation patterning. We propose that these genes contribute to regulate vein patterning, likely controlling auxin homeostasis.

A role for LAX2 in regulating xylem development and lateral-vein symmetry in the leaf.

Background and Aims: The symmetry of venation patterning in leaves is highly conserved within a plant species. Auxins are involved in this process and also in xylem vasculature development. Studying transgenic Arabidopsis plants ectopically expressing the sunflower transcription factor HaHB4, it was observed that there was a significant lateral-vein asymmetry in leaves and in xylem formation compared to wild type plants. To unravel the molecular mechanisms behind this phenotype, genes differentially expressed in these plants and related to auxin influx were investigated. Methods: Candidate genes responsible for the observed phenotypes were selected using a co-expression analysis. Single and multiple mutants in auxin influx carriers were characterized by morphological, physiological and molecular techniques. The analysis was further complemented by restoring the wild type (WT) phenotype by mutant complementation studies and using transgenic soybean plants ectopically expressing HaHB4 . Key Results: LAX2 , down-regulated in HaHB4 transgenic plants, was bioinformatically chosen as a candidate gene. The quadruple mutant aux1 lax1 lax2 lax3 and the single mutants, except lax1, presented an enhanced asymmetry in venation patterning. Additionally, the xylem vasculature of the lax2 mutant and the HaHB4 -expressing plants differed from the WT vasculature, including increased xylem length and number of xylem cell rows. Complementation of the lax2 mutant with the LAX2 gene restored both lateral-vein symmetry and xylem/stem area ratio in the stem, showing that auxin homeostasis is required to achieve normal vascular development. Interestingly, soybean plants ectopically expressing HaHB4 also showed an increased asymmetry in the venation patterning, accompanied by the repression of several GmLAX genes. Conclusions: Auxin influx carriers have a significant role in leaf venation pattering in leaves and, in particular, LAX2 is required for normal xylem development, probablt controlling auxin homeostasis.

Phytochrome regulation of plant immunity in vegetation canopies.

Plant immunity against pathogens and herbivores is a central determinant of plant fitness in nature and crop yield in agroecosystems. Plant immune responses are orchestrated by two key hormones: jasmonic acid (JA) and salicylic acid (SA). Recent work has demonstrated that for plants of shade-intolerant species, which include the majority of those grown as grain crops, light is a major modulator of defense responses. Light signals that indicate proximity of competitors, such as a low red to far-red (R:FR) ratio, down-regulate the expression of JA- and SA-induced immune responses against pests and pathogens. This down-regulation of defense under low R:FR ratios, which is caused by the photoconversion of the photoreceptor phytochrome B (phyB) to an inactive state, is likely to help the plant to efficiently redirect resources to rapid growth when the competition threat posed by neighboring plants is high. This review is focused on the molecular mechanisms that link phyB with defense signaling. In particular, we discuss novel signaling players that are likely to play a role in the repression of defense responses under low R:FR ratios. A better understanding of the molecular connections between photoreceptors and the hormonal regulation of plant immunity will provide a functional framework to understand the mechanisms used by plants to deal with fundamental resource allocation trade-offs under dynamic conditions of biotic stress.

The flavonoid biosynthetic enzyme chalcone isomerase modulates terpenoid production in glandular trichomes of tomato.

Flavonoids and terpenoids are derived from distinct metabolic pathways but nevertheless serve complementary roles in mediating plant interactions with the environment. Here, we show that glandular trichomes of the anthocyanin free (af) mutant of cultivated tomato (Solanum lycopersicum) fail to accumulate both flavonoids and terpenoids. This pleiotropic metabolic deficiency was associated with loss of resistance to native populations of coleopteran herbivores under field conditions. We demonstrate that Af encodes an isoform (SlCHI1) of the flavonoid biosynthetic enzyme chalcone isomerase (CHI), which catalyzes the conversion of naringenin chalcone to naringenin and is strictly required for flavonoid production in multiple tissues of tomato. Expression of the wild-type SlCHI1 gene from its native promoter complemented the anthocyanin deficiency in af. Unexpectedly, the SlCHI1 transgene also complemented the defect in terpenoid production in glandular trichomes. Our results establish a key role for SlCHI1 in flavonoid production in tomato and reveal a link between CHI1 and terpenoid production. Metabolic coordination of the flavonoid and terpenoid pathways may serve to optimize the function of trichome glands in dynamic environments.

A bHLH-type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1, acts as a repressor to negatively regulate jasmonate signaling in arabidopsis.

Jasmonates (JAs) are plant hormones that regulate the balance between plant growth and responses to biotic and abiotic stresses. Although recent studies have uncovered the mechanisms for JA-induced responses in Arabidopsis thaliana, the mechanisms by which plants attenuate the JA-induced responses remain elusive. Here, we report that a basic helix-loop-helix-type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1 (JAM1), acts as a transcriptional repressor and negatively regulates JA signaling. Gain-of-function transgenic plants expressing the chimeric repressor for JAM1 exhibited substantial reduction of JA responses, including JA-induced inhibition of root growth, accumulation of anthocyanin, and male fertility. These plants were also compromised in resistance to attack by the insect herbivore Spodoptera exigua. Conversely, jam1 loss-of-function mutants showed enhanced JA responsiveness, including increased resistance to insect attack. JAM1 and MYC2 competitively bind to the target sequence of MYC2, which likely provides the mechanism for negative regulation of JA signaling and suppression of MYC2 functions by JAM1. These results indicate that JAM1 negatively regulates JA signaling, thereby playing a pivotal role in fine-tuning of JA-mediated stress responses and plant growth.

Negative feedback control of jasmonate signaling by an alternative splice variant of JAZ10.

The plant hormone jasmonate (JA) activates gene expression by promoting ubiquitin-dependent degradation of jasmonate ZIM domain (JAZ) transcriptional repressor proteins. A key feature of all JAZ proteins is the highly conserved Jas motif, which mediates both JAZ degradation and JAZ binding to the transcription factor MYC2. Rapid expression of JAZ genes in response to JA is thought to attenuate JA responses, but little is known about the mechanisms by which newly synthesized JAZ proteins exert repression in the presence of the hormone. Here, we show in Arabidopsis (Arabidopsis thaliana) that desensitization to JA is mediated by an alternative splice variant (JAZ10.4) of JAZ10 that lacks the Jas motif. Unbiased protein-protein interaction screens identified three related basic helix-loop-helix transcription factors (MYC2, MYC3, and MYC4) and the corepressor NINJA as JAZ10.4-binding partners. We show that the amino-terminal region of JAZ10.4 contains a cryptic MYC2-binding site that resembles the Jas motif and that the ZIM motif of JAZ10.4 functions as a transferable repressor domain whose activity is associated with the recruitment of NINJA. Functional studies showed that the expression of JAZ10.4 from the native JAZ10 promoter complemented the JA-hypersensitive phenotype of a jaz10 mutant. Moreover, treatment of these complemented lines with JA resulted in the rapid accumulation of JAZ10.4 protein. Our results provide an explanation for how the unique domain architecture of JAZ10.4 links transcription factors to a corepressor complex and suggest how JA-induced transcription and alternative splicing of JAZ10 premessenger RNA creates a regulatory circuit to attenuate JA responses.

JAZ8 lacks a canonical degron and has an EAR motif that mediates transcriptional repression of jasmonate responses in Arabidopsis.

The lipid-derived hormone jasmonoyl-L-Ile (JA-Ile) initiates large-scale changes in gene expression by stabilizing the interaction of JASMONATE ZIM domain (JAZ) repressors with the F-box protein CORONATINE INSENSITIVE1 (COI1), which results in JAZ degradation by the ubiquitin-proteasome pathway. Recent structural studies show that the JAZ1 degradation signal (degron) includes a short conserved LPIAR motif that seals JA-Ile in its binding pocket at the COI1-JAZ interface. Here, we show that Arabidopsis thaliana JAZ8 lacks this motif and thus is unable to associate strongly with COI1 in the presence of JA-Ile. As a consequence, JAZ8 is stabilized against jasmonate (JA)-mediated degradation and, when ectopically expressed in Arabidopsis, represses JA-regulated growth and defense responses. These findings indicate that sequence variation in a hypervariable region of the degron affects JAZ stability and JA-regulated physiological responses. We also show that JAZ8-mediated repression depends on an LxLxL-type EAR (for ERF-associated amphiphilic repression) motif at the JAZ8 N terminus that binds the corepressor TOPLESS and represses transcriptional activation. JAZ8-mediated repression does not require the ZIM domain, which, in other JAZ proteins, recruits TOPLESS through the EAR motif-containing adaptor protein NINJA. These findings show that EAR repression domains in a subgroup of JAZ proteins repress gene expression through direct recruitment of corepressors to cognate transcription factors.

Cryptochrome 1 and phytochrome B control shade-avoidance responses in Arabidopsis via partially independent hormonal cascades.

Plants respond to a reduction in the red/far-red ratio (R:FR) of light, caused by the proximity of other plants, by initiating morphological changes that improve light capture. In Arabidopsis, this response (shade avoidance syndrome, SAS) is controlled by phytochromes (particularly phyB), and is dependent on the TAA1 pathway of auxin biosynthesis. However, when grown in real canopies, we found that phyB mutants and mutants deficient in TAAI (sav3) still display robust SAS responses to increased planting density and leaf shading. The SAS morphology (leaf hyponasty and reduced lamina/petiole ratio) could be phenocopied by exposing plants to blue light attenuation. These responses to blue light attenuation required the UV-A/blue light photoreceptor cry1. Moreover, they were mediated through mechanisms that showed only limited overlap with the pathways recruited by phyB inactivation. In particular, pathways for polar auxin transport, auxin biosynthesis and gibberellin signaling that are involved in SAS responses to low R:FR were not required for the SAS responses to blue light depletion. By contrast, the brassinosteroid response appeared to be required for the full expression of the SAS phenotype under low blue light. The phyB and cry1 inactivation pathways appeared to converge in their requirement for the basic/helix-loop-helix (bHLH) transcription factors PHYTOCHROME INTERACTING FACTORs 4 and 5 (PIF4 and PIF5) to elicit the SAS phenotype. Our results suggest that blue light is an important control of SAS responses, and that PIF4 and PIF5 are critical hubs for a diverse array of signaling routes that control plant architecture in canopies.

Phytochrome B enhances photosynthesis at the expense of water-use efficiency in Arabidopsis.

In open places, plants are exposed to higher fluence rates of photosynthetically active radiation and to higher red to far-red ratios than under the shade of neighbor plants. High fluence rates are known to increase stomata density. Here we show that high, compared to low, red to far-red ratios also increase stomata density in Arabidopsis (Arabidopsis thaliana). High red to far-red ratios increase the proportion of phytochrome B (phyB) in its active form and the phyB mutant exhibited a constitutively low stomata density. phyB increased the stomata index (the ratio between stomata and epidermal cells number) and the level of anphistomy (by increasing stomata density more intensively in the adaxial than in the abaxial face). phyB promoted the expression of FAMA and TOO MANY MOUTHS genes involved in the regulation of stomata development in young leaves. Increased stomata density resulted in increased transpiration per unit leaf area. However, phyB promoted photosynthesis rates only at high fluence rates of photosynthetically active radiation. In accordance to these observations, phyB reduced long-term water-use efficiency estimated by the analysis of isotopic discrimination against (13)CO(2). We propose a model where active phyB promotes stomata differentiation in open places, allowing plants to take advantage of the higher irradiances at the expense of a reduction of water-use efficiency, which is compensated by a reduced leaf area.

Ecological modulation of plant defense via phytochrome control of jasmonate sensitivity.

For plants, the tradeoff between resource investment in defense and increased growth to out-compete neighbors creates an allocation dilemma. How plants resolve this dilemma, at the mechanistic level, is unclear. We found that Arabidopsis plants produced an attenuated defense phenotype under conditions of crowding and when exposed to far-red (FR) radiation, a light signal that plants use to detect the proximity of neighbors via the photoreceptor phytochrome. This phenotype was detectable through standard bioassays that measured the growth of Spodoptera frugiperda caterpillars. Two possible explanations for the effect of FR are: (i) a simple by-product of the diversion of resources to competition, and (ii) a specific effect of phytochrome on defense signaling. The first possibility was ruled out by the fact that the auxin-deficient sav3 mutant, which fails to induce growth responses to FR, still responded to FR with an attenuated defense phenotype. In support of the second hypothesis, we found that phytochrome inactivation by FR caused a strong reduction of plant sensitivity to jasmonates, which are key regulators of plant immunity. The effects of FR on jasmonate sensitivity were restricted to certain elements of the pathway. Supporting the idea that the FR effects on jasmonate signaling are functionally significant, we found that FR failed to increase tissue quality in jar1, a mutant impaired in jasmonate response. We conclude that the plant modulates its investment in defense as a function of the perceived risk of competition, and that this modulation is effected by phytochrome via selective desensitization to jasmonates.

Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants.

Plants grown at high densities perceive a decrease in the red to far-red (R:FR) ratio of incoming light, resulting from absorption of red light by canopy leaves and reflection of far-red light from neighboring plants. These changes in light quality trigger a series of responses known collectively as the shade avoidance syndrome. During shade avoidance, stems elongate at the expense of leaf and storage organ expansion, branching is inhibited, and flowering is accelerated. We identified several loci in Arabidopsis, mutations in which lead to plants defective in multiple shade avoidance responses. Here we describe TAA1, an aminotransferase, and show that TAA1 catalyzes the formation of indole-3-pyruvic acid (IPA) from L-tryptophan (L-Trp), the first step in a previously proposed, but uncharacterized, auxin biosynthetic pathway. This pathway is rapidly deployed to synthesize auxin at the high levels required to initiate the multiple changes in body plan associated with shade avoidance.