Knock-Down of Arabidopsis PLC5 Reduces Primary Root Growth and Secondary Root Formation While Overexpression Improves Drought Tolerance and Causes Stunted Root Hair Growth.

Phospholipase C (PLC) is a well-known signaling enzyme in metazoans that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate and diacylglycerol as second messengers involved in mutiple processes. Plants contain PLC too, but relatively little is known about its function there. The model system Arabidopsis thaliana contains nine PLC genes. Reversed genetics have implicated several roles for PLCs in plant development and stress signaling. Here, PLC5 is functionally addressed. Promoter-ß-glucuronidase (GUS) analyses revealed expression in roots, leaves and flowers, predominantly in vascular tissue, most probably phloem companion cells, but also in guard cells, trichomes and root apical meristem. Only one plc5-1 knock-down mutant was obtained, which developed normally but grew more slowly and exhibited reduced primary root growth and decreased lateral root numbers. These phenotypes could be complemented by expressing the wild-type gene behind its own promoter. Overexpression of PLC5 (PLC5-OE) using the UBQ10 promoter resulted in reduced primary and secondary root growth, stunted root hairs, decreased stomatal aperture and improved drought tolerance. PLC5-OE lines exhibited strongly reduced phosphatidylinositol 4-monophosphate (PIP) and PIP2 levels and increased amounts of phosphatidic acid, indicating enhanced PLC activity in vivo. Reduced PIP2 levels and stunted root hair growth of PLC5-OE seedlings could be recovered by inducible overexpression of a root hair-specific PIP 5-kinase, PIP5K3. Our results show that PLC5 is involved in primary and secondary root growth and that its overexpression improves drought tolerance. Independently, we provide new evidence that PIP2 is essential for the polar tip growth of root hairs.

Arabidopsis Phospholipase C3 is Involved in Lateral Root Initiation and ABA Responses in Seed Germination and Stomatal Closure.

Phospholipase C (PLC) is well known for its role in animal signaling, where it generates the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), by hydrolyzing the minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), upon receptor stimulation. In plants, PLC's role is still unclear, especially because the primary targets of both second messengers are lacking, i.e. the ligand-gated Ca2+ channel and protein kinase C, and because PIP2 levels are extremely low. Nonetheless, the Arabidopsis genome encodes nine PLCs. We used a reversed-genetic approach to explore PLC's function in Arabidopsis, and report here that PLC3 is required for proper root development, seed germination and stomatal opening. Two independent knock-down mutants, plc3-2 and plc3-3, were found to exhibit reduced lateral root densities by 10-20%. Mutant seeds germinated more slowly but were less sensitive to ABA to prevent germination. Guard cells of plc3 were also compromised in ABA-dependent stomatal closure. Promoter-ß-glucuronidase (GUS) analyses confirmed PLC3 expression in guard cells and germinating seeds, and revealed that the majority is expressed in vascular tissue, most probably phloem companion cells, in roots, leaves and flowers. In vivo 32Pi labeling revealed that ABA stimulated the formation of PIP2 in germinating seeds and guard cell-enriched leaf peels, which was significantly reduced in plc3 mutants. Overexpression of PLC3 had no effect on root system architecture or seed germination, but increased the plant's tolerance to drought. Our results provide genetic evidence for PLC's involvement in plant development and ABA signaling, and confirm earlier observations that overexpression increases drought tolerance. Potential molecular mechanisms for the above observations are discussed.

Long-Distance Lipid Signaling and its Role in Plant Development and Stress Response.

Lipids are important signaling compounds in plants. They can range from small lipophilic molecules like the dicarboxylic acid Azelaic acid to complex phosphoglycerolipids and regulate plant development as well as the response to biotic and abiotic stress. While their intracellular function is well described, several lipophilic signals are known to be found in the plant phloem and can, thus, also play a role in long-distance signaling. Mostly, they play a role in the pathogen response and systemic acquired resistance. This is particularly true for oxylipins, dehydroabietinal, and azelaic acid. However, several phospholipids have now been described in phloem exudates. Their intracellular function as well as implications and a model for long-distance signaling are discussed in this chapter.

Arabidopsis acyl-CoA-binding protein ACBP6 localizes in the phloem and affects jasmonate composition.

Arabidopsis thaliana ACYL-COA-BINDING PROTEIN6 (AtACBP6) encodes a cytosolic 10-kDa AtACBP. It confers freezing tolerance in transgenic Arabidopsis, possibly by its interaction with lipids as indicated by the binding of acyl-CoA esters and phosphatidylcholine to recombinant AtACBP6. Herein, transgenic Arabidopsis transformed with an AtACBP6 promoter-driven ß-glucuronidase (GUS) construct exhibited strong GUS activity in the vascular tissues. Immunoelectron microscopy using anti-AtACBP6 antibodies showed AtACBP6 localization in the phloem especially in the companion cells and sieve elements. Also, the presence of gold grains in the plasmodesmata indicated its potential role in systemic trafficking. The AtACBP6 protein, but not its mRNA, was found in phloem exudate of wild-type Arabidopsis. Fatty acid profiling using gas chromatography-mass spectrometry revealed an increase in the jasmonic acid (JA) precursor, 12-oxo-cis,cis-10,15-phytodienoic acid (cis-OPDA), and a reduction in JA and/or its derivatives in acbp6 phloem exudates in comparison to the wild type. Quantitative real-time PCR showed down-regulation of COMATOSE (CTS) in acbp6 rosettes suggesting that AtACBP6 affects CTS function. AtACBP6 appeared to affect the content of JA and/or its derivatives in the sieve tubes, which is consistent with its role in pathogen-defense and in its wound-inducibility of AtACBP6pro::GUS. Taken together, our results suggest the involvement of AtACBP6 in JA-biosynthesis in Arabidopsis phloem tissues.

PLAT domain protein 1 (PLAT1/PLAFP) binds to the Arabidopsis thaliana plasma membrane and inserts a lipid.

Robust agricultural yields depend on the plant's ability to fix carbon amid variable environmental conditions. Over seasonal and diurnal cycles, the plant must constantly adjust its metabolism according to available resources or external stressors. The metabolic changes that a plant undergoes in response to stress are well understood, but the long-distance signaling mechanisms that facilitate communication throughout the plant are less studied. The phloem is considered the predominant conduit for the bidirectional transport of these signals in the form of metabolites, nucleic acids, proteins, and lipids. Lipid trafficking through the phloem in particular attracted our attention due to its reliance on soluble lipid-binding proteins (LBP) that generate and solubilize otherwise membrane-associated lipids. The Phloem Lipid-Associated Family Protein (PLAFP) from Arabidopsis thaliana is generated in response to abiotic stress as is its lipid-ligand phosphatidic acid (PA). PLAFP is proposed to transport PA through the phloem in response to drought stress. To understand the interactions between PLAFP and PA, nearly 100 independent systems comprised of the protein and one PA, or a plasma membrane containing varying amounts of PA, were simulated using atomistic classical molecular dynamics methods. In these simulations, PLAFP is found to bind to plant plasma membrane models independent of the PA concentration. When bound to the membrane, PLAFP adopts a binding pose where W41 and R82 penetrate the membrane surface and anchor PLAFP. This triggers a separation of the two loop regions containing W41 and R82. Subsequent simulations indicate that PA insert into the ß-sandwich of PLAFP, driven by interactions with multiple amino acids besides the W41 and R82 identified during the insertion process. Fine-tuning the protein-membrane and protein-PA interface by mutating a selection of these amino acids may facilitate engineering plant signaling processes by modulating the binding response.

Contribution of proteomics in the identification of novel proteins associated with plant growth.

The epidermis is not only the interphase between the plant and the environment but also a growth-limiting tissue. Understanding the initiation and regulation of its expansion growth is essential for addressing the need for more food and fuel. We used mass spectrometry to identify proteins from auxin (indole-3-acetic acid; IAA)-induced rapidly growing corn (Zea mays) coleoptiles to find possible candidates controlling this growth as well as the underlying cell wall and cuticle biosynthesis. Excised sections were incubated for 4 h in the absence or presence of IAA, protein extracted, and analyzed using LC-ESI-MS/MS. Of 86 proteins identified, 15 showed a predicted association with cell wall/cuticle biosynthesis or trafficking machinery; four identifications revealed novel proteins of unknown function. In parallel, real-time PCR indicated that the steady-state mRNA levels of genes with a known or predicted role in cell-wall biosynthesis increase upon treatment with auxin. Importantly, genes encoding two of the hypothetical proteins also show higher levels of mRNA; additionally, their gene expression is down-regulated as coleoptile growth ceases and up-regulated in expanding leaves. This suggests a major role of those novel proteins in the regulation of processes related to cell and organ expansion and thus plant growth.

Identification of lipids and lipid-binding proteins in phloem exudates from Arabidopsis thaliana.

The phloem plays a crucial role in assimilate and nutrient transport, pathogen response, and plant growth and development. Yet, few species have yielded pure phloem exudate and, if proteins need to be analysed, those species may not have sequenced genomes, making identification difficult. The enrichment of Arabidopsis thaliana phloem exudate in amounts large enough to allow for metabolite and protein analysis is described. Using this method, it was possible to identify 65 proteins present in the Arabidopsis phloem exudate. The majority of these proteins could be grouped by response to pathogens, stress, or hormones, carbon metabolism, protein interaction, modification, and turnover, and transcription factors. It was also possible to detect 11 proteins that play a role in lipid/fatty acid metabolism (aspartic protease, putative 3-ß-hydroxysteroid dehydrogenase, UDP-sulphoquinovose synthase/SQD1, lipase, PIG-P-like protein: phosphatidylinositol-N-acetylglucosaminyltransferase), storage (glycine-rich protein), binding (annexin, lipid-associated family protein, GRP17/oleosin), and/or signalling (annexin, putative lipase, PIG-P-like protein). Along with putative lipid-binding proteins, several lipids and fatty acids could be identified. Only a few examples exist of lipids (jasmonic acid, oxylipins) or lipid-binding proteins (DIR1, acyl-CoA-binding protein) in the phloem. Finding hydrophobic compounds in an aqueous environment is not without precedence in biological systems: human blood contains a variety of lipids, many of which play a significant role in human health. In blood, lipids are transported while bound to proteins. The present findings of lipids and lipid-binding proteins in phloem exudates suggest that a similar long-distance lipid signalling exists in plants and may play an important role in plant growth and development.

Collection and Analysis of Phloem Lipids.

The plant phloem is a long-distance conduit for the transport of assimilates but also of mobile developmental and stress signals. These signals can be sugars, metabolites, amino acids, peptides, proteins, microRNA, or mRNA. Yet small lipophilic molecules such as oxylipins and, more recently, phospholipids have emerged as possible long-distance signals as well. Analysis of phloem (phospho)lipids, however, requires enrichment, purification, and sensitive analysis. This chapter describes the EDTA-facilitated approach of phloem exudate collection, phase partitioning against chloroform-methanol for lipid separation and enrichment, and analysis/identification of phloem lipids using LC-MS with multiplexed collision induced dissociation (CID).

The interplay of phloem-mobile signals in plant development and stress response.

Plants integrate a variety of biotic and abiotic factors for optimal growth in their given environment. While some of these responses are local, others occur distally. Hence, communication of signals perceived in one organ to a second, distal part of the plant and the coordinated developmental response require an intricate signaling system. To do so, plants developed a bipartite vascular system that mediates the uptake of water, minerals, and nutrients from the soil; transports high-energy compounds and building blocks; and traffics essential developmental and stress signals. One component of the plant vasculature is the phloem. The development of highly sensitive mass spectrometry and molecular methods in the last decades has enabled us to explore the full complexity of the phloem content. As a result, our view of the phloem has evolved from a simple transport path of photoassimilates to a major highway for pathogens, hormones and developmental signals. Understanding phloem transport is essential to comprehend the coordination of environmental inputs with plant development and, thus, ensure food security. This review discusses recent developments in its role in long-distance signaling and highlights the role of some of the signaling molecules. What emerges is an image of signaling paths that do not just involve single molecules but rather, quite frequently an interplay of several distinct molecular classes, many of which appear to be transported and acting in concert.

Phloem Proteomics Reveals New Lipid-Binding Proteins with a Putative Role in Lipid-Mediated Signaling.

Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three proteins are indeed lipid-binding and act in the vasculature possibly in a function related to long-distance signaling, the three proteins do not act in the same but rather in distinct pathways. It also points toward PLAFP as a prime candidate to investigate long-distance lipid signaling in the plant drought response.

Proteomic analysis of changes in the Kandelia candel chloroplast proteins reveals pathways associated with salt tolerance.

The plant chloroplast is one of the most sensitive organelles in response to salt stress. Chloroplast proteins extracted from seedling leaves were separated by two-dimensional gel electrophoresis (2-DE). More than 600 protein spots could be distinguished on each gel. Fifty-eight differentially expressed protein spots were detected, of which 46 could be identified through matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). These proteins were found to be involved in multiple aspects of chloroplast metabolism pathways such as photosynthesis, ATP synthesis, detoxification and antioxidation processes, nitrogen assimilation and fixation, protein metabolism, and tetrapyrrole biosynthesis. The results indicated that K. candel could withstand up to 500 mM NaCl stress for a measured period of 3 days, by maintaining normal or high photosynthetic electron transfer efficiency and an only slightly stimulated Calvin cycle. Meanwhile, we found that ROS scavenging, nitrogen assimilation, protein degradation and chaperone function in chloroplasts were also of importance for salt tolerance of K. candel. The ultrastructural and physiological data agree with chloroplast proteome results. These findings allow further exploration of our knowledge on salt adaptation in woody halophytes and may contribute to the development of more salt-tolerant plants in the future.

Collection and analysis of Arabidopsis phloem exudates using the EDTA-facilitated Method.

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Comparative proteomics of chloroplasts envelopes from bundle sheath and mesophyll chloroplasts reveals novel membrane proteins with a possible role in c4-related metabolite fluxes and development.

As the world population grows, our need for food increases drastically. Limited amounts of arable land lead to a competition between food and fuel crops, while changes in the global climate may impact future crop yields. Thus, a second "green revolution" will need a better understanding of the processes essential for plant growth and development. One approach toward the solution of this problem is to better understand regulatory and transport processes in C4 plants. C4 plants display an up to 10-fold higher apparent CO2 assimilation and higher yields while maintaining high water use efficiency. This requires differential regulation of mesophyll (M) and bundle sheath (BS) chloroplast development as well as higher metabolic fluxes of photosynthetic intermediates between cells and particularly across chloroplast envelopes. While previous analyses of overall chloroplast membranes have yielded significant insight, our comparative proteomics approach using enriched BS and M chloroplast envelopes of Zea mays allowed us to identify 37 proteins of unknown function that have not been seen in these earlier studies. We identified 280 proteins, 84% of which are known/predicted to be present in chloroplasts. Seventy-four percent have a known or predicted membrane association. Twenty-one membrane proteins were 2-15 times more abundant in BS cells, while 36 of the proteins were more abundant in M chloroplast envelopes. These proteins could represent additional candidates of proteins essential for development or metabolite transport processes in C4 plants. RT-PCR confirmed differential expression of 13 candidate genes. Chloroplast association for seven proteins was confirmed using YFP/GFP labeling. Gene expression of four putative transporters was examined throughout the leaf and during the greening of leaves. Genes for a PIC-like protein and an ER-AP-like protein show an early transient increase in gene expression during the transition to light. In addition, PIC gene expression is increased in the immature part of the leaf and was lower in the fully developed parts of the leaf, suggesting a need for/incorporation of the protein during chloroplast development.

New aspects of Phloem-mediated long-distance lipid signaling in plants.

Plants are sessile and cannot move to appropriate hiding places or feeding grounds to escape adverse conditions. As a consequence, they evolved mechanisms to detect changes in their environment, communicate these to different organs, and adjust development accordingly. These adaptations include two long-distance transport systems which are essential in plants: the xylem and the phloem. The phloem serves as a major trafficking pathway for assimilates, viruses, RNA, plant hormones, metabolites, and proteins with functions ranging from synthesis to metabolism to signaling. The study of signaling compounds within the phloem is essential for our understanding of plant communication of environmental cues. Determining the nature of signals and the mechanisms by which they are communicated through the phloem will lead to a more complete understanding of plant development and plant responses to stress. In our analysis of Arabidopsis phloem exudates, we had identified several lipid-binding proteins as well as fatty acids and lipids. The latter are not typically expected in the aqueous environment of sieve elements. Hence, lipid transport in the phloem has been given little attention until now. Long-distance transport of hydrophobic compounds in an aqueous system is not without precedence in biological systems: a variety of lipids is found in human blood and is often bound to proteins. Some lipid-protein complexes are transported to other tissues for storage, use, modification, or degradation; others serve as messengers and modulate transcription factor activity. By simple analogy it raises the possibility that lipids and the respective lipid-binding proteins in the phloem serve similar functions in plants and play an important role in stress and developmental signaling. Here, we introduce the lipid-binding proteins and the lipids we found in the phloem and discuss the possibility that they may play an important role in developmental and stress signaling.

Comparative proteomics of chloroplast envelopes from C3 and C4 plants reveals specific adaptations of the plastid envelope to C4 photosynthesis and candidate proteins required for maintaining C4 metabolite fluxes.

C(4) plants have up to 10-fold higher apparent CO(2) assimilation rates than the most productive C(3) plants. This requires higher fluxes of metabolic intermediates across the chloroplast envelope membranes of C(4) plants in comparison with those of C(3) plants. In particular, the fluxes of metabolites involved in the biochemical inorganic carbon pump of C(4) plants, such as malate, pyruvate, oxaloacetate, and phosphoenolpyruvate, must be considerably higher in C(4) plants because they exceed the apparent rate of photosynthetic CO(2) assimilation, whereas they represent relatively minor fluxes in C(3) plants. While the enzymatic steps involved in the C(4) biochemical inorganic carbon pump have been studied in much detail, little is known about the metabolite transporters in the envelope membranes of C(4) chloroplasts. In this study, we used comparative proteomics of chloroplast envelope membranes from the C(3) plant pea (Pisum sativum) and mesophyll cell chloroplast envelopes from the C(4) plant maize (Zea mays) to analyze the adaptation of the mesophyll cell chloroplast envelope proteome to the requirements of C(4) photosynthesis. We show that C(3)- and C(4)-type chloroplasts have qualitatively similar but quantitatively very different chloroplast envelope membrane proteomes. In particular, translocators involved in the transport of triosephosphate and phosphoenolpyruvate as well as two outer envelope porins are much more abundant in C(4) plants. Several putative transport proteins have been identified that are highly abundant in C(4) plants but relatively minor in C(3) envelopes. These represent prime candidates for the transport of C(4) photosynthetic intermediates, such as pyruvate, oxaloacetate, and malate.

The protein kinase CK2 phosphorylates SNAP190 to negatively regulate SNAPC DNA binding and human U6 transcription by RNA polymerase III.

Human U6 small nuclear RNA gene transcription by RNA polymerase III requires the general transcription factor SNAP(C), which binds to human small nuclear RNA core promoter elements and nucleates pre-initiation complex assembly with the Brf2-TFIIIB complex. Multiple components in this pathway are phosphorylated by the protein kinase CK2, including the Bdp1 subunit of the Brf2-TFIIIB complex, and RNA polymerase III, with negative and positive outcomes for U6 transcription, respectively. However, a role for CK2 phosphorylation of SNAP(C) in U6 transcription has not been defined. In this report, we investigated the role of CK2 in modulating the transcriptional properties of SNAP(C) and demonstrate that within SNAP(C), CK2 phosphorylates the N-terminal half of the SNAP190 subunit at two regions (amino acids 20-63 and 514-545) that each contain multiple CK2 consensus sites. SNAP190 phosphorylation by CK2 inhibits both SNAP(C) DNA binding and U6 transcription activity. Mutational analyses of SNAP190 support a model wherein CK2 phosphorylation triggers an allosteric inhibition of the SNAP190 Myb DNA binding domain.

WRI1 is required for seed germination and seedling establishment.

Storage compound accumulation during seed development prepares the next generation of plants for survival. Therefore, processes involved in the regulation and synthesis of storage compound accumulation during seed development bear relevance to germination and seedling establishment. The wrinkled1 (wri1) mutant of Arabidopsis (Arabidopsis thaliana) is impaired in seed oil accumulation. The WRI1 gene encodes an APETALA2/ethylene-responsive element-binding protein transcription factor involved in the control of metabolism, particularly glycolysis, in the developing seeds. Here we investigate the role of this regulatory factor in seed germination and seedling establishment by comparing the wri1-1 mutant, transgenic lines expressing the WRI1 wild-type cDNA in the wri1-1 mutant background, and the wild type. Plants altered in the expression of the WRI1 gene showed different germination responses to the growth factor abscisic acid (ABA), sugars, and fatty acids provided in the medium. Germination of the mutant was more sensitive to ABA, sugars, and osmolites, an effect that was alleviated by increased WRI1 expression in transgenic lines. The expression of ABA-responsive genes AtEM6 and ABA-insensitive 3 (ABI3) was increased in the wri1-1 mutant. Double-mutant analysis between abi3-3 and wri1-1 suggested that WRI1 and ABI3, a transcription factor mediating ABA responses in seeds, act in parallel pathways. Addition of 2-deoxyglucose inhibited seed germination, but did so less in lines overexpressing WRI1. Seedling establishment was decreased in the wri1-1 mutant but could be alleviated by sucrose. Apart from a possible signaling role in germination, sugars in the medium were required as building blocks and energy supply during wri1-1 seedling establishment.