The CELL NUMBER REGULATOR FW2.2 protein regulates cell-to-cell communication in tomato by modulating callose deposition at plasmodesmata.

FW2.2 (standing for FRUIT WEIGHT 2.2), the founding member of the CELL NUMBER REGULATOR (CNR) gene family, was the first cloned gene underlying a quantitative trait locus (QTL) governing fruit size and weight in tomato (Solanum lycopersicum). However, despite this discovery over 20 yr ago, the molecular mechanisms by which FW2.2 negatively regulates cell division during fruit growth remain undeciphered. In the present study, we confirmed that FW2.2 is a membrane-anchored protein whose N- and C-terminal ends face the apoplast. We unexpectedly found that FW2.2 is located at plasmodesmata (PD). FW2.2 participates in the spatiotemporal regulation of callose deposition at PD and belongs to a protein complex which encompasses callose synthases. These results suggest that FW2.2 has a regulatory role in cell-to-cell communication by modulating PD transport capacity and trafficking of signaling molecules during fruit development.

Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.

Iron-sulfur (Fe-S) clusters are one of the most ancient and ubiquitous prosthetic groups, and they are required by a variety of proteins involved in important metabolic processes. Apicomplexan parasites have inherited different plastidic and mitochondrial Fe-S clusters biosynthesis pathways through endosymbiosis. We have investigated the relative contributions of these pathways to the fitness of Toxoplasma gondii, an apicomplexan parasite causing disease in humans, by generating specific mutants. Phenotypic analysis and quantitative proteomics allowed us to highlight notable differences in these mutants. Both Fe-S cluster synthesis pathways are necessary for optimal parasite growth in vitro, but their disruption leads to markedly different fates: impairment of the plastidic pathway leads to a loss of the organelle and to parasite death, while disruption of the mitochondrial pathway trigger differentiation into a stress resistance stage. This highlights that otherwise similar biochemical pathways hosted by different sub-cellular compartments can have very different contributions to the biology of the parasites, which is something to consider when exploring novel strategies for therapeutic intervention.

The Transcription Factor bHLH121 Interacts with bHLH105 (ILR3) and Its Closest Homologs to Regulate Iron Homeostasis in Arabidopsis.

Iron (Fe) is an essential micronutrient for plant growth and development. Any defects in the maintenance of Fe homeostasis will alter plant productivity and the quality of their derived products. In Arabidopsis (Arabidopsis thaliana), the transcription factor ILR3 plays a central role in controlling Fe homeostasis. In this study, we identified bHLH121 as an ILR3-interacting transcription factor. Interaction studies showed that bHLH121 also interacts with the three closest homologs of ILR3 (i.e., basic-helix-loop-helix 34 [bHLH34], bHLH104, and bHLH115). bhlh121 loss-of-function mutants displayed severe defects in Fe homeostasis that could be reverted by exogenous Fe supply. bHLH121 acts as a direct transcriptional activator of key genes involved in the Fe regulatory network, including bHLH38, bHLH39, bHLH100, bHLH101, POPEYE, BRUTUS, and BRUTUS LIKE1, as well as IRONMAN1 and IRONMAN2 In addition, bHLH121 is necessary for activating the expression of transcription factor gene FIT in response to Fe deficiency via an indirect mechanism. bHLH121 is expressed throughout the plant body, and its expression is not affected by Fe availability. By contrast, Fe availability affects the cellular localization of bHLH121 protein in roots. Altogether, these data show that bHLH121 is a regulator of Fe homeostasis that acts upstream of FIT in concert with ILR3 and its closest homologs.

Protein abundance in the midgut of wild tsetse flies (Glossina palpalis palpalis) naturally infected by Trypanosoma congolense s.l.

Tsetse flies (Glossina spp.) are major vectors of African trypanosomes, causing either Human or Animal African Trypanosomiasis (HAT or AAT). Several approaches have been developed to control the disease, among which is the anti-vector Sterile Insect Technique. Another approach to anti-vector strategies could consist of controlling the fly's vector competence through hitherto unidentified regulatory factors (genes, proteins, biological pathways, etc.). The present work aims to evaluate the protein abundance in the midgut of wild tsetse flies (Glossina palpalis palpalis) naturally infected by Trypanosoma congolense s.l. Infected and non-infected flies were sampled in two HAT/AAT foci in Southern Cameroon. After dissection, the proteomes from the guts of parasite-infected flies were compared to that of uninfected flies to identify quantitative and/or qualitative changes associated with infection. Among the proteins with increased abundance were fructose-1,6-biphosphatase, membrane trafficking proteins, death proteins (or apoptosis proteins) and SERPINs (inhibitor of serine proteases, enzymes considered as trypanosome virulence factors) that displayed the highest increased abundance. The present study, together with previous proteomic and transcriptomic studies on the secretome of trypanosomes from tsetse fly gut extracts, provides data to be explored in further investigations on, for example, mammal host immunisation or on fly vector competence modification via para-transgenic approaches.

Root Membrane Ubiquitinome under Short-Term Osmotic Stress.

Osmotic stress can be detrimental to plants, whose survival relies heavily on proteomic plasticity. Protein ubiquitination is a central post-translational modification in osmotic-mediated stress. In this study, we used the K-Ɛ-GG antibody enrichment method integrated with high-resolution mass spectrometry to compile a list of 719 ubiquitinated lysine (K-Ub) residues from 450 Arabidopsis root membrane proteins (58% of which are transmembrane proteins), thereby adding to the database of ubiquitinated substrates in plants. Although no ubiquitin (Ub) motifs could be identified, the presence of acidic residues close to K-Ub was revealed. Our ubiquitinome analysis pointed to a broad role of ubiquitination in the internalization and sorting of cargo proteins. Moreover, the simultaneous proteome and ubiquitinome quantification showed that ubiquitination is mostly not involved in membrane protein degradation in response to short osmotic treatment but that it is putatively involved in protein internalization, as described for the aquaporin PIP2;1. Our in silico analysis of ubiquitinated proteins shows that two E2 Ub-conjugating enzymes, UBC32 and UBC34, putatively target membrane proteins under osmotic stress. Finally, we revealed a positive role for UBC32 and UBC34 in primary root growth under osmotic stress.

Coumarin accumulation and trafficking in Arabidopsis thaliana: a complex and dynamic process.

Iron (Fe) is a major micronutrient and is required for plant growth and development. Nongrass species have evolved a reduction-based strategy to solubilize and take up Fe. The secretion of Fe-mobilizing coumarins (e.g. fraxetin, esculetin and sideretin) by plant roots plays an important role in this process. Although the biochemical mechanisms leading to their biosynthesis have been well described, very little is known about their cellular and subcellular localization or their mobility within plant tissues. Spectral imaging was used to monitor, in Arabidopsis thaliana, the in planta localization of Fe-mobilizing coumarins and scopolin. Molecular, genetic and biochemical approaches were also used to investigate the dynamics of coumarin accumulation in roots. These approaches showed that root hairs play a major role in scopoletin secretion, whereas fraxetin and esculetin secretion occurs through all epidermis cells. The findings of this study also showed that the transport of coumarins from the cortex to the rhizosphere relies on the PDR9 transporter under Fe-deficient conditions. Additional experiments support the idea that coumarins move throughout the plant body via the xylem sap and that several plant species can take up coumarins present in the surrounding media. Altogether, the data presented here demonstrate that coumarin storage and accumulation in roots is a highly complex and dynamic process.

Protein lysine methylation contributes to modulating the response of sensitive and tolerant Arabidopsis species to cadmium stress.

The mechanisms underlying the response and adaptation of plants to excess of trace elements are not fully described. Here, we analysed the importance of protein lysine methylation for plants to cope with cadmium. We analysed the effect of cadmium on lysine-methylated proteins and protein lysine methyltransferases (KMTs) in two cadmium-sensitive species, Arabidopsis thaliana and A. lyrata, and in three populations of A. halleri with contrasting cadmium accumulation and tolerance traits. We showed that some proteins are differentially methylated at lysine residues in response to Cd and that a few genes coding KMTs are regulated by cadmium. Also, we showed that 9 out of 23 A. thaliana mutants disrupted in KMT genes have a tolerance to cadmium that is significantly different from that of wild-type seedlings. We further characterized two of these mutants, one was knocked out in the calmodulin lysine methyltransferase gene and displayed increased tolerance to cadmium, and the other was interrupted in a KMT gene of unknown function and showed a decreased capacity to cope with cadmium. Together, our results showed that lysine methylation of non-histone proteins is impacted by cadmium and that several methylation events are important for modulating the response of Arabidopsis plants to cadmium stress.

Identification of client iron-sulfur proteins of the chloroplastic NFU2 transfer protein in Arabidopsis thaliana.

Iron-sulfur (Fe-S) proteins have critical functions in plastids, notably participating in photosynthetic electron transfer, sulfur and nitrogen assimilation, chlorophyll metabolism, and vitamin or amino acid biosynthesis. Their maturation relies on the so-called SUF (sulfur mobilization) assembly machinery. Fe-S clusters are synthesized de novo on a scaffold protein complex and then delivered to client proteins via several transfer proteins. However, the maturation pathways of most client proteins and their specificities for transfer proteins are mostly unknown. In order to decipher the proteins interacting with the Fe-S cluster transfer protein NFU2, one of the three plastidial representatives found in Arabidopsis thaliana, we performed a quantitative proteomic analysis of shoots, roots, and seedlings of nfu2 plants, combined with NFU2 co-immunoprecipitation and binary yeast two-hybrid experiments. We identified 14 new targets, among which nine were validated in planta using a binary bimolecular fluorescence complementation assay. These analyses also revealed a possible role for NFU2 in the plant response to desiccation. Altogether, this study better delineates the maturation pathways of many chloroplast Fe-S proteins, considerably extending the number of NFU2 clients. It also helps to clarify the respective roles of the three NFU paralogs NFU1, NFU2, and NFU3.

A Global Proteomic Approach Sheds New Light on Potential Iron-Sulfur Client Proteins of the Chloroplastic Maturation Factor NFU3.

Iron-sulfur (Fe-S) proteins play critical functions in plants. Most Fe-S proteins are synthetized in the cytosol as apo-proteins and the subsequent Fe-S cluster incorporation relies on specific protein assembly machineries. They are notably formed by a scaffold complex, which serves for the de novo Fe-S cluster synthesis, and by transfer proteins that insure cluster delivery to apo-targets. However, scarce information is available about the maturation pathways of most plastidial Fe-S proteins and their specificities towards transfer proteins of the associated SUF machinery. To gain more insights into these steps, the expression and protein localization of the NFU1, NFU2, and NFU3 transfer proteins were analyzed in various Arabidopsis thaliana organs and tissues showing quite similar expression patterns. In addition, quantitative proteomic analysis of an nfu3 loss-of-function mutant allowed to propose novel potential client proteins for NFU3 and to show that the protein accumulation profiles and thus metabolic adjustments differ substantially from those established in the nfu2 mutant. By clarifying the respective roles of the three plastidial NFU paralogs, these data allow better delineating the maturation process of plastidial Fe-S proteins.

Regulation of a plant aquaporin by a Casparian strip membrane domain protein-like.

The absorption of soil water by roots allows plants to maintain their water status. At the endodermis, water transport can be affected by initial formation of a Casparian strip and further deposition of suberin lamellas and regulated by the function of aquaporins. Four Casparian strip membrane domain protein-like (CASPL; CASPL1B1, CASPL1B2, CASPL1D1, and CASPL1D2) were previously shown to interact with PIP2;1. The present work shows that CASPL1B1, CASPL1B2, and CASPL1D2 are exclusively expressed in suberized endodermal cells, suggesting a cell-specific role in suberization and/or water transport regulation. When compared with wild-type plants, and by contrast to caspl1b1*caspl1b2 double loss of function, caspl1d1*caspl1d2 double mutants showed, in some control or NaCl stress experiments and not upon abscisic acid (ABA) treatment, a weak enlargement of the continuous suberization zone. None of the mutants showed root hydraulic conductivity (Lpr ) phenotype, whether in control, NaCl, or ABA treatment conditions. The data suggest a slight negative role for CASPL1D1 and CASPL1D2 in suberization under control or salt stress conditions, with no major impact on whole root transport functions. At the molecular level, CASPL1B1 was able to physically interact with PIP2;1 and potentially could influence the regulation of aquaporins by acting on their phosphorylated form.

Integrative analysis of the late maturation programme and desiccation tolerance mechanisms in intermediate coffee seeds.

The 'intermediate seed' category was defined in the early 1990s using coffee (Coffea arabica) as a model. In contrast to orthodox seeds, intermediate seeds cannot survive complete drying, which is a major constraint for seed storage and has implications for both biodiversity conservation and agricultural purposes. However, intermediate seeds are considerably more tolerant to drying than recalcitrant seeds, which are highly sensitive to desiccation. To gain insight into the mechanisms governing such differences, changes in desiccation tolerance (DT), hormone contents, and the transcriptome were analysed in developing coffee seeds. Acquisition of DT coincided with a dramatic transcriptional switch characterised by the repression of primary metabolism, photosynthesis, and respiration, and the up-regulation of genes coding for late-embryogenesis abundant (LEA) proteins, heat-shock proteins (HSPs), and antioxidant enzymes. Analysis of the heat-stable proteome in mature coffee seeds confirmed the accumulation of LEA proteins identified at the transcript level. Transcriptome analysis also suggested a major role for ABA and for the transcription factors CaHSFA9, CaDREB2G, CaANAC029, CaPLATZ, and CaDOG-like in DT acquisition. The ability of CaHSFA9 and CaDREB2G to trigger HSP gene transcription was validated by Agrobacterium-mediated transformation of coffee somatic embryos.

Novel Aquaporin Regulatory Mechanisms Revealed by Interactomics.

PIP1;2 and PIP2;1 are aquaporins that are highly expressed in roots and bring a major contribution to root water transport and its regulation by hormonal and abiotic factors. Interactions between cellular proteins or with other macromolecules contribute to forming molecular machines. Proteins that molecularly interact with PIP1;2 and PIP2;1 were searched to get new insights into regulatory mechanisms of root water transport. For that, a immuno-purification strategy coupled to protein identification and quantification by mass spectrometry (IP-MS) of PIPs was combined with data from the literature, to build thorough PIP1;2 and PIP2;1 interactomes, sharing about 400 interacting proteins. Such interactome revealed PIPs to behave as a platform for recruitment of a wide range of transport activities and provided novel insights into regulation of PIP cellular trafficking by osmotic and oxidative treatments. This work also pointed a role of lipid signaling in PIP function and enhanced our knowledge of protein kinases involved in PIP regulation. In particular we show that 2 members of the receptor-like kinase (RLK) family (RKL1 (At1g48480) and Feronia (At3g51550)) differentially modulate PIP activity through distinct molecular mechanisms. The overall work opens novel perspectives in understanding PIP regulatory mechanisms and their role in adjustment of plant water status.

FUBP1: a new protagonist in splicing regulation of the DMD gene.

We investigated the molecular mechanisms for in-frame skipping of DMD exon 39 caused by the nonsense c.5480T>A mutation in a patient with Becker muscular dystrophy. RNase-assisted pull down assay coupled with mass spectrometry revealed that the mutant RNA probe specifically recruits hnRNPA1, hnRNPA2/B1 and DAZAP1. Functional studies in a human myoblast cell line transfected with DMD minigenes confirmed the splicing inhibitory activity of hnRNPA1 and hnRNPA2/B1, and showed that DAZAP1, also known to activate splicing, acts negatively in the context of the mutated exon 39. Furthermore, we uncovered that recognition of endogenous DMD exon 39 in muscle cells is promoted by FUSE binding protein 1 (FUBP1), a multifunctional DNA- and RNA-binding protein whose role in splicing is largely unknown. By serial deletion and mutagenesis studies in minigenes, we delineated a functional intronic splicing enhancer (ISE) in intron 38. FUBP1 recruitment to the RNA sequence containing the ISE was established by RNA pull down and RNA EMSA, and further confirmed by RNA-ChIP on endogenous DMD pre-mRNA. This study provides new insights about the splicing regulation of DMD exon 39, highlighting the emerging role of FUBP1 in splicing and describing the first ISE for constitutive exon inclusion in the mature DMD transcript.

Convergent Evolution of Endosymbiont Differentiation in Dalbergioid and Inverted Repeat-Lacking Clade Legumes Mediated by Nodule-Specific Cysteine-Rich Peptides.

Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen-fixing symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly within highly specialized symbiotic host cells. In Inverted Repeat-Lacking Clade (IRLC) legumes such as Medicago spp., the bacteroids are kept under control by an arsenal of nodule-specific cysteine-rich (NCR) peptides, which induce the bacteria in an irreversible, strongly elongated, and polyploid state. Here, we show that in Aeschynomene spp. legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical depending on the Aeschynomene spp. and that these bacteroids are terminally differentiated and polyploid, similar to bacteroids in IRLC legumes. Transcriptome, in situ hybridization, and proteome analyses demonstrated that the symbiotic cells in the Aeschynomene spp. nodules produce a large diversity of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNA interference-mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved independently from the bacteroid differentiation in the IRLC clade, is based on very similar mechanisms used by IRLC legumes.

Histone H3 serine-57 is a CHK1 substrate whose phosphorylation affects DNA repair.

Histone post-translational modifications promote a chromatin environment that controls transcription, DNA replication and repair, but surprisingly few phosphorylations have been documented. We report the discovery of histone H3 serine-57 phosphorylation (H3S57ph) and show that it is implicated in different DNA repair pathways from fungi to vertebrates. We identified CHK1 as a major human H3S57 kinase, and disrupting or constitutively mimicking H3S57ph had opposing effects on rate of recovery from replication stress, 53BP1 chromatin binding, and dependency on RAD52. In fission yeast, mutation of all H3 alleles to S57A abrogated DNA repair by both non-homologous end-joining and homologous recombination, while cells with phospho-mimicking S57D alleles were partly compromised for both repair pathways, presented aberrant Rad52 foci and were strongly sensitised to replication stress. Mechanistically, H3S57ph loosens DNA-histone contacts, increasing nucleosome mobility, and interacts with H3K56. Our results suggest that dynamic phosphorylation of H3S57 is required for DNA repair and recovery from replication stress, opening avenues for investigating the role of this modification in other DNA-related processes.

Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves.

BACKGROUND: The N-terminal proline-rich domain (Zera) of the maize storage protein γ-zein, is able to induce the formation of endoplasmic reticulum (ER)-derived protein bodies (PBs) when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. The aim of the present work was to evaluate if induced PBs encapsulate additional proteins jointly with the recombinant protein. The exhaustive analysis of protein composition of PBs is expected to facilitate a better understanding of PB formation and the optimization of recombinant protein purification approaches from these organelles. RESULTS: We analysed the proteome of PBs induced in Nicotiana benthamiana leaves by transient transformation with Zera fused to a fluorescent marker protein (DsRed). Intact PBs with their surrounding ER-membrane were isolated on iodixanol based density gradients and their integrity verified by confocal and electron microscopy. SDS-PAGE analysis of isolated PBs showed that Zera-DsRed accounted for around 85% of PB proteins in term of abundance. Differential extraction of PBs was performed for in-depth analysis of their proteome and structure. Besides Zera-DsRed, 195 additional proteins were identified including a broad range of proteins resident or trafficking through the ER and recruited within the Zera-DsRed polymer. CONCLUSIONS: This study indicates that Zera-protein fusion is still the major protein component of the new formed organelle in tobacco leaves. The analysis also reveals the presence of an unexpected diversity of proteins in PBs derived from both the insoluble Zera-DsRed polymer formation, including ER-resident and secretory proteins, and a secretory stress response induced most likely by the recombinant protein overloading. Knowledge of PBs protein composition is likely to be useful to optimize downstream purification of recombinant proteins in molecular farming applications.

Phosphoproteomic analysis reveals major default phosphorylation sites outside long intrinsically disordered regions of Arabidopsis plasma membrane proteins.

BACKGROUND: Genome-wide statistics established that long intrinsically disordered regions (over 30 residues) are predicted in a large part of proteins in all eukaryotes, with a higher ratio in trans-membrane proteins. At functional level, such unstructured and flexible regions were suggested for years to favour phosphorylation events. In plants, despite increasing evidence of the regulation of transport and signalling processes by phosphorylation events, only few data are available without specific information regarding plasma membrane proteins, especially at proteome scale. RESULTS: Using a dedicated phosphoproteomic workflow, 75 novel and unambiguous phosphorylation sites were identified in Arabidopsis plasma membrane. Bioinformatics analysis showed that this new dataset concerned mostly integral proteins involved in key functions of the plasma membrane (such as transport and signal transduction, including protein phosphorylation). It thus expanded by 15% the directory of phosphosites previously characterized in signalling and transport proteins. Unexpectedly, 66% of phosphorylation sites were predicted to be located outside long intrinsically disordered regions. This result was further corroborated by analysis of publicly available data for the plasma membrane. CONCLUSIONS: The new phosphoproteomics data presented here, with published datasets and functional annotation, suggest a previously unexpected topology of phosphorylation in the plant plasma membrane proteins. The significance of these new insights into the so far overlooked properties of the plant plasma membrane phosphoproteome and the long disordered regions is discussed.

Excreted/secreted proteins from trypanosome procyclic strains.

Trypanosoma secretome was shown to be involved in parasite virulence and is suspected of interfering in parasite life-cycle steps such as establishment in the Glossina midgut, metacyclogenesis. Therefore, we attempted to identify the proteins secreted by procyclic strains of T. brucei gambiense and T. brucei brucei, responsible for human and animal trypanosomiasis, respectively. Using mass spectrometry, 427 and 483 nonredundant proteins were characterized in T. brucei brucei and T. brucei gambiense secretomes, respectively; 35% and 42% of the corresponding secretome proteins were specifically secreted by T. brucei brucei and T. brucei gambiense, respectively, while 279 proteins were common to both subspecies. The proteins were assigned to 12 functional classes. Special attention was paid to the most abundant proteases (14 families) because of their potential implication in the infection process and nutrient supply. The presence of proteins usually secreted via an exosome pathway suggests that this type of process is involved in trypanosome ESP secretion. The overall results provide leads for further research to develop novel tools for blocking trypanosome transmission.

Targeted Proteomics Allows Quantification of Ethylene Receptors and Reveals SlETR3 Accumulation in Never-Ripe Tomatoes.

Ethylene regulates fruit ripening and several plant functions (germination, plant growth, plant-microbe interactions). Protein quantification of ethylene receptors (ETRs) is essential to study their functions, but is impaired by low resolution tools such as antibodies that are mostly nonspecific, or the lack of sensitivity of shotgun proteomic approaches. We developed a targeted proteomic method, to quantify low-abundance proteins such as ETRs, and coupled this to mRNAs analyses, in two tomato lines: Wild Type (WT) and Never-Ripe (NR) which is insensitive to ethylene because of a gain-of-function mutation in ETR3. We obtained mRNA and protein abundance profiles for each ETR over the fruit development period. Despite a limiting number of replicates, we propose Pearson correlations between mRNA and protein profiles as interesting indicators to discriminate the two genotypes: such correlations are mostly positive in the WT and are affected by the NR mutation. The influence of putative post-transcriptional and post-translational changes are discussed. In NR fruits, the observed accumulation of the mutated ETR3 protein between ripening stages (Mature Green and Breaker + 8 days) may be a cause of NR tomatoes to stay orange. The label-free quantitative proteomics analysis of membrane proteins, concomitant to Parallel Reaction Monitoring analysis, may be a resource to study changes over tomato fruit development. These results could lead to studies about ETR subfunctions and interconnections over fruit development. Variations of RNA-protein correlations may open new fields of research in ETR regulation. Finally, similar approaches may be developed to study ETRs in whole plant development and plant-microorganism interactions.

Visible and fluorescent staining of two-dimensional gels.

Staining of two-dimensional gel constitutes a crucial step in comparative proteome analysis with respect to the number of proteins analyzed, the accuracy of spot quantification, and compatibility with mass spectrometry. The present chapter describes procedures for several visible and fluorescent dyes compatible with mass spectrometry: colloidal Coomassie blue, silver nitrate, Sypro Ruby, Deep Purple, and 5-hexadecanoylamino-fluorescein.