YUCCA2 (YUC2)-Mediated 3-Indoleacetic Acid (IAA) Biosynthesis Regulates Chloroplast RNA Editing by Relieving the Auxin Response Factor 1 (ARF1)-Dependent Inhibition of Editing Factors in Arabidopsis thaliana.

Although recent research progress on the abundant C-to-U RNA editing events in plant chloroplasts and mitochondria has uncovered many recognition factors and their molecular mechanisms, the intrinsic regulation of RNA editing within plants remains largely unknown. This study aimed to establish a regulatory relationship in Arabidopsis between the plant hormone auxin and chloroplast RNA editing. We first analyzed auxin response elements (AuxREs) present within promoters of chloroplast editing factors reported to date. We found that each has more than one AuxRE, suggesting a potential regulatory role of auxin in their expression. Further investigation unveiled that the depletion of auxin synthesis gene YUC2 reduces the expression of several editing factors. However, in yuc2 mutants, only the expression of CRR4, DYW1, ISE2, and ECD1 editing factors and the editing efficiency of their corresponding editing sites, ndhD-2 and rps14-149, were simultaneously suppressed. In addition, exogenous IAA and the overexpression of YUC2 enhanced the expression of these editing factors and the editing efficiency at the ndhD-2 and rps14-149 sites. These results suggested a direct effect of auxin upon the editing of the ndhD-2 and rps14-149 sites through the modulation of the expression of the editing factors. We further demonstrated that ARF1, a downstream transcription factor in the auxin-signaling pathway, could directly bind to and inactivate the promoters of CRR4, DYW1, and ISE2 in a dual-luciferase reporter system, thereby inhibiting their expression. Moreover, the overexpression of ARF1 in Arabidopsis significantly reduced the expression of the three editing factors and the editing efficiency at the ndhD-2 and rps14-149 sites. These data suggest that YUC2-mediated auxin biosynthesis governs the RNA-editing process through the ARF1-dependent signal transduction pathway.

Auxin inhibits chlorophyll accumulation through ARF7-IAA14-mediated repression of chlorophyll biosynthesis genes in Arabidopsis.

Auxin is a well-known important phytohormone in plant that plays vital roles in almost every development process throughout plant lifecycle. However, the effect of auxin on the metabolism of chlorophyll, one of the most important pigments involved in the photosynthesis, was intertwined and the underlying mechanism remained to be explored. Here, we found the auxin-defective yuc2 yuc6 double mutant displayed dark-green leaf color with higher chlorophyll content than wildtype, suggesting a negative regulatory role of auxin in chlorophyll biosynthesis. The chloroplast number and structure in mesophyll cells were altered and the photosynthetic efficiency was improved in yuc2 yuc6. In addition, the chlorophyll level was significantly improved during seedling de-etiolation in yuc2 yuc6 mutant, and decreased dramatically under IAA treatment, confirming the inhibitory role of auxin in chlorophyll biosynthesis. The analyses of gene expression in mature leaves and de-etiolation seedlings suggested that auxin suppressed the expression of many chlorophyll biosynthesis genes, especially PROTOCHLOROPHYLLIDE OXIDOREDUCTASE A (PORA) and GENOMES UNCOUPLED 5 (GUN5). Yeast-one-hybrid and luciferase assays demonstrated that the AUXIN RESPONSE FACTOR 2 (ARF2) and ARF7 bind to the promoter of PORA and GUN5 to suppress their expression with the help of INDOLE-3-ACETIC ACID14 (IAA14). Collectively, our research explicitly unraveled the direct inhibitory role of auxin in chlorophyll biosynthesis, and provided new insight into the interplay between auxin signaling and chlorophyll metabolism.

The PPR protein RARE1-mediated editing of chloroplast accD transcripts is required for fatty acid biosynthesis and heat tolerance in Arabidopsis.

It has been reported that Arabidopsis chloroplast accD transcripts undergo RNA editing and that loss of accD-C794 RNA editing does not affect plant growth under normal conditions. To date, the exact biological role of accD-C794 editing has remained elusive. Here, we reveal an unexpected role for accD-C794 editing in response to heat stress. Loss of accD-C794 editing results in a yellow and dwarf phenotype with decreased chloroplast gene expression under heat stress, and artificial improvement of C794-edited accD gene expression enhances heat tolerance in Arabidopsis. These data suggest that accD-C794 editing confers heat tolerance in planta. We also found that treatment with the product of acetyl coenzyme A carboxylase (ACCase) could allay mutant phenotypic characteristics and showed that a mutation in the CAC3 gene for the α-subunit of ACCase was associated with dwarfism under heat stress. These observations indicate that defective accD-C794 editing may be intrinsic to reduced ACCase activity, thereby contributing to heat sensitivity. ACCase catalyzes the committed step of de novo fatty acid (FA) biosynthesis. FA content analysis revealed that unsaturated oleic (C18:1) and linoleic acids (C18:2) were low in the accD-C794 editing-defective mutant but high in the C794-edited accD-overexpressing plants compared with the wild type. Supplying exogenous C18:1 and C18:2 could rescue the mutant phenotype, suggesting that these FAs play an essential role in tolerance to heat stress. Transmission electron microscopy observations showed that heat stress seriously affected the membrane architecture in accD editing-defective mutants but not in accD-overexpressing plants. These results provide the first evidence that accD-C794 editing regulates FA biosynthesis for maintenance of membrane structural homeostasis under heat stress.

A mass spectrometry imaging approach on spatiotemporal distribution of multiple alkaloids in Gelsemium elegans.

Gelsemium elegans contains multiple alkaloids with pharmacological effects, thus researchers focus on the identification and application of alkaloids extracted from G. elegans. Regretfully, the spatiotemporal distribution of alkaloids in G. elegans is still unclear. In this study, the desorption electrospray ionization mass spectrometry imaging (DESI-MSI) was applied to simultaneously analyze the distribution of pharmacologically important alkaloids in different organ/tissue sections of G. elegans at different growth stages. Finally, 23 alkaloids were visualized in roots, stems and leaves at seedling stage and 19 alkaloids were observed at mature stage. In mature G. elegans, 16 alkaloids were distributed in vascular bundle region of mature roots, 15 alkaloids were mainly located in the pith region of mature stems and 2 alkaloids were enriched in epidermis region of mature stems. A total of 16 alkaloids were detected in leaf veins of mature leaves and 17 alkaloids were detected in shoots. Interestingly, diffusion and transfer of multiple alkaloids in tissues have been observed along with the development and maturation. This study comprehensively characterized the spatial metabolomics of G. elegans alkaloids, and the spatiotemporal distribution of alkaloid synthesis. In addition, the results also have reference value for the development and application of Gelsemium elegans and other medicinal plants.

In Situ Visual Distribution of Gelsemine, Koumine, and Gelsenicine by MSI in Gelsemiumelegans at Different Growth Stages.

The distribution of pharmatically important alkaloids gelsemine, koumine, and gelsenicine in Gelsemium elegans tissues is a hot topic attracting research attention. Regretfully, the in planta visual distribution details of these alkaloids are far from clear although several researches reported the alkaloid quantification in G. elegans by LC-MS/MS. In this study, mass imaging spectrometry (MSI) was employed to visualize the in situ visualization of gelsemine, koumine, and gelsenicine in different organs and tissues of G. elegans at different growth stages, and the relative quantification of three alkaloids were performed according to the image brightness intensities captured by the desorption electrospray ionization MSI (DESI-MSI). The results indicated that these alkaloids were mainly accumulated in pith region and gradually decreased from pith to epidermis. Interestingly, three alkaloids were found to be present in higher abundance in the leaf vein. Along with the growth and development, the accumulation of these alkaloids was gradually increased in root and stem. Moreover, we employed LC-MS/MS to quantify three alkaloids and further validated the in situ distributions. The content of koumine reached 249.2 µg/g in mature roots, 272.0 µg/g in mature leaves, and 149.1 µg/g in mature stems, respectively, which is significantly higher than that of gelsemine and gelsenicine in the same organ. This study provided an accurately in situ visualization of gelsemine, koumine, and gelsenicine in G. elegans, and would be helpful for understanding their accumulation in plant and guiding application.

3D Visualization and Volume-Based Quantification of Rice Chalkiness In Vivo by Using High Resolution Micro-CT.

BACKGROUND: Rice quality research attracts attention worldwide. Rice chalkiness is one of the key indexes determining rice kernel quality. The traditional rice chalkiness measurement methods only use milled rice as materials and are mainly based on naked-eye observation or area-based two-dimensional (2D) image analysis and the results could not represent the three-dimensional (3D) characteristics of chalkiness in the rice kernel. These methods are neither in vivo thus are unable to analyze living rice seeds for high throughput screening of rice chalkiness phenotype. RESULTS: Here, we introduced a novel method for 3D visualization and accurate volume-based quantification of rice chalkiness in vivo by using X-ray microcomputed tomography (micro-CT). This approach not only develops a novel volume-based method to measure the 3D rice chalkiness index, but also provides a high throughput solution for rice chalkiness phenotype analysis by using living rice seeds. CONCLUSIONS: Our method could be a new powerful tool for rice chalkiness measurement, especially for high throughput chalkiness phenotype screening using living rice seeds. This method could be used in chalkiness phenotype identification and screening, and would greatly promote the basic research in rice chalkiness regulation as well as the quality evaluation in rice production practice.

Phytohormone dynamics in developing endosperm influence rice grain shape and quality.

Hormones are important signaling molecules regulating developmental processes and responses to environmental stimuli in higher plants. Rice endosperm, the portion of the seed surrounding the embryo, is the main determinant of rice grain shape and yield; however, the dynamics and exact functions of phytohormones in developing endosperm remain elusive. Through a systemic study including transcriptome analysis, hormone measurement, and transgene-based endosperm-specific expression of phytohormone biosynthetic enzymes, we demonstrated that dynamic phytohormone levels play crucial roles in the developing rice endosperm, particularly in regard to grain shape and quality. We detected diverse, differential, and dramatically changing expression patterns of genes related to hormone biosynthesis and signaling during endosperm development, especially at early developmental stages. Liquid chromatography measurements confirmed the dynamic accumulation of hormones in developing endosperm. Further transgenic analysis performed on plants expressing hormone biosynthesis genes driven by an endosperm-specific promoter revealed differential effects of the hormones, especially auxin and brassinosteroids, in regulating grain shape and quality. Our studies help elucidate the distinct roles of hormones in developing endosperm and provide novel and useful tools for influencing crop seed shape and yield.

IPyA glucosylation mediates light and temperature signaling to regulate auxin-dependent hypocotyl elongation in Arabidopsis.

Auxin is a class of plant hormone that plays a crucial role in the life cycle of plants, particularly in the growth response of plants to ever-changing environments. Since the auxin responses are concentration-dependent and higher auxin concentrations might often be inhibitory, the optimal endogenous auxin level must be closely controlled. However, the underlying mechanism governing auxin homeostasis remains largely unknown. In this study, a UDP-glycosyltransferase (UGT76F1) was identified from Arabidopsis thaliana, which participates in the regulation of auxin homeostasis by glucosylation of indole-3-pyruvic acid (IPyA), a major precursor of the auxin indole-3-acetic acid (IAA) biosynthesis, in the formation of IPyA glucose conjugates (IPyA-Glc). In addition, UGT76F1 was found to mediate hypocotyl growth by modulating active auxin levels in a light- and temperature-dependent manner. Moreover, the transcription of UGT76F1 was demonstrated to be directly and negatively regulated by PIF4, which is a key integrator of both light and temperature signaling pathways. This study sheds a light on the trade-off between IAA biosynthesis and IPyA-Glc formation in controlling auxin levels and reveals a regulatory mechanism for plant growth adaptation to environmental changes through glucosylation of IPyA.

Dynamic Cytology and Transcriptional Regulation of Rice Lamina Joint Development.

Rice (Oryza sativa) leaf angle is determined by lamina joint and is an important agricultural trait determining leaf erectness and, hence, the photosynthesis efficiency and grain yield. Genetic studies reveal a complex regulatory network of lamina joint development; however, the morphological changes, cytological transitions, and underlying transcriptional programming remain to be elucidated. A systemic morphological and cytological study reveals a dynamic developmental process and suggests a common but distinct regulation of the lamina joint. Successive and sequential cell division and expansion, cell wall thickening, and programmed cell death at the adaxial or abaxial sides form the cytological basis of the lamina joint, and the increased leaf angle results from the asymmetric cell proliferation and elongation. Analysis of the gene expression profiles at four distinct developmental stages ranging from initiation to senescence showed that genes related to cell division and growth, hormone synthesis and signaling, transcription (transcription factors), and protein phosphorylation (protein kinases) exhibit distinct spatiotemporal patterns during lamina joint development. Phytohormones play crucial roles by promoting cell differentiation and growth at early stages or regulating the maturation and senescence at later stages, which is consistent with the quantitative analysis of hormones at different stages. Further comparison with the gene expression profile of leaf inclination1, a mutant with decreased auxin and increased leaf angle, indicates the coordinated effects of hormones in regulating lamina joint. These results reveal a dynamic cytology of rice lamina joint that is fine-regulated by multiple factors, providing informative clues for illustrating the regulatory mechanisms of leaf angle and plant architecture.

Plant hormone jasmonate prioritizes defense over growth by interfering with gibberellin signaling cascade.

Plants must effectively defend against biotic and abiotic stresses to survive in nature. However, this defense is costly and is often accompanied by significant growth inhibition. How plants coordinate the fluctuating growth-defense dynamics is not well understood and remains a fundamental question. Jasmonate (JA) and gibberellic acid (GA) are important plant hormones that mediate defense and growth, respectively. Binding of bioactive JA or GA ligands to cognate receptors leads to proteasome-dependent degradation of specific transcriptional repressors (the JAZ or DELLA family of proteins), which, at the resting state, represses cognate transcription factors involved in defense (e.g., MYCs) or growth [e.g. phytochrome interacting factors (PIFs)]. In this study, we found that the coi1 JA receptor mutants of rice (a domesticated monocot crop) and Arabidopsis (a model dicot plant) both exhibit hallmark phenotypes of GA-hypersensitive mutants. JA delays GA-mediated DELLA protein degradation, and the della mutant is less sensitive to JA for growth inhibition. Overexpression of a selected group of JAZ repressors in Arabidopsis plants partially phenocopies GA-associated phenotypes of the coi1 mutant, and JAZ9 inhibits RGA (a DELLA protein) interaction with transcription factor PIF3. Importantly, the pif quadruple (pifq) mutant no longer responds to JA-induced growth inhibition, and overexpression of PIF3 could partially overcome JA-induced growth inhibition. Thus, a molecular cascade involving the COI1-JAZ-DELLA-PIF signaling module, by which angiosperm plants prioritize JA-mediated defense over growth, has been elucidated.

[Analysis on the characters of progenies of the rice transformed with anti-sense waxy gene].

The segregation of exogenous genes was studied by hygromycin-resistant and PCR experiments in the transgenic rice (Oryza sativa L. subsp. japonica and indica) with anti-sense waxy gene, meanwhile the change of amylose and waxy protein contents in progenies of transgenic rice was analyzed. The results showed no matter the rice Guangjing No.1 (O. Sativa L. subsp. japonica) were transformed by p13w4 plasmid carrying anti-sense waxy gene and hygromycin-resistant gene, or in the rice 01Z5202 (O. sativa L. subsp. indica) were co-transformed by p13w8 plasmid carrying anti-sense waxy gene and p1300 plasmid carrying hygromycin-resistant gene, the target gene(s) had been segregated in the progenies; the content of amylose of the transgenic plants was lower than those in non-transgenic ones, and the content of amylose in some of transgenic plants was less than 10.0% (of the weight of grain), which was much lower than those in the control (about 22.04%); and the analysis with SDS-PAGE showed the content of the waxy protein are positively correlated with the con-tent of amylose in the tested transgenic rice materials.

An impedance immunosensor for the detection of the phytohormone abscisic acid.

The phytohormone abscisic acid (ABA) is the major player in mediating the adaptation of plants to stress. Previously developed phytohormonal biosensors usually employed indirect detection of the products of conjugated oxidase reactions. A label-free electrochemical impedance immunosensor for ABA detection was developed using an anti-ABA antibody adsorbed directly on a porous nanogold film. The film was produced electrochemically on a glassy carbon electrode in 0.008 mol/L hydrogen tetrachloroaurate solution containing 0.004 mol/L lead acetate with an applied potential of -0.5 V (versus Ag/AgCl) for 50 s. The anti-ABA antibody was immobilized onto the porous nanogold through electrostatic adsorption and covalent conjugation. Electrochemical impedance spectroscopy was used to characterize the successful construction of the porous nanogold film and the stepwise modification of the glassy carbon electrode. The concentration increase of the antigen brought about a decrease of the interfacial electron transfer, which also meant an increase of the impedance signal. The experimental parameters pH, antibody incubation time, and antibody concentration were optimized. The results showed significant linearity R = 0.9942, with the content of ABA in the range 0.5-5,000 ng/mL with a detection limit at about 0.1 ng/mL.

Arabidopsis replication factor C subunit 1 plays an important role in embryogenesis.

Replication factor C (RFC), consisting of one large subunit and four small subunits, is an important factor involved in DNA replication and repair mechanisms as well as cell proliferation. The subunit 1 of Arabidopsis RFC (AtRFC1) is a homologue of p140, the large subunit of human RFC. Three T-DNA insertion mutant lines of AtRFC1, i.e. rfc1-1, rfc1-2 and rfc1-3, with insertion mutations located in exons 16 and 19, and the promoter region respectively were verified. These mutations caused defects in embryogenesis and led to embryo and seed abortion. Transformation of wild type AtRFC1 gene into rfc1 mutant alleles reverted the mutant phenotypes, suggesting that AtRFC1 plays an important role in embryo development in Arabidopsis thaliana.

Photosynthesis of the flag leaf blade and its sheath in high-yielding hybrid rice 'Liangyoupeijiu'.

Using high-yielding hybrid rice 'Liangyoupeijiu' (LYP9) and hybrid rice 'Shanyou 63' (SY63) as the experimental materials and using (14)C radio-autography, the photosynthetic capacities and distribution of photosynthates in flag leaf blades and sheaths of LYP9 were studied. The results showed that net photosynthetic rates (Pn) of the flag leaf blades and sheaths of LYP9 were much higher than those of SY63; the light transmissivity rates (LT) measured at the medium height of the flag leaf sheaths and the penultimate leaf sheaths were also significantly higher than those of SY63. The incipient activities, total activities and activation percentages of Rubisco in the flag leaf blade and sheath of LYP9 were all higher than those of SY63. The photosynthate transport rate in the sheaths of LYP9, and the quantity of photosynthate transported to the spikes and transformed to economic yield of LYP9 were all higher than those of SY63. The photosynthates produced by the sheaths were mainly transported to spike to make a certain contribution (about 15%) to yield.

[Effects of Cd2+ on seedling growth and phytohormone contents of Glycine max].

A laboratory incubation experiment was conducted to study the effects of different Cd2+ concentrations on seedling growth and phytohormone contents of Glycine max through determining some physiological and biochemical indexes. The results showed as follows: (1) Different Cd2+ concentrations inhibited the synthesis of indoleacetic acid (IAA) and gibberellins (GA3) in roots and stimulated the synthesis of zeatin (Z) and abscisic acid (ABA) not only in roots but also in aerial parts of Glycine Max. Cd2+ stimulated the synthesis of IAA and GA3 in aerial parts at lower concentrations, but inhibited the synthesis of IAA and GA3 at higher concentrations. (2) Cd2+ stress for 84h increased the root vitality and the contents of chlorophyll a and b, but no significant difference was found in carotinoid contents compared with the control. (3) Cd2+ stress decreased POD activities and MAD contents atlower concentrations and increased POD activities and MAD contents at higher concentrations. With increasing Cd2+ concentrations, this increase became significant gradually. (4) Cd2+ stress with lower concentrations stimulated the physiological activity of Glycine max seedling in a short time. When Cd2+ concentration was 0.50 mg/L, the root vitality, IAA and GA3 contents of Glycine max were the highest, and POD activity was the lowest.

[Changes in polyamine levels in Citrus sinensis Osb. cv. Valencia callus during somatic embryogenesis].

Somatic embryogenetic capability and changes in polyamine level and their relationship were analyzed using the long-term (8 years) subcultured calli of Citrus sinensis Osb. cv. Valencia as materials. The results showed that endogenous polyamine contents in embryogenic calli were higher than those in non-embryogenic calli, and the embryogenetic capability was positively correlated to the levels of endogenous polyamines. When the calli were transferred to a differentiation medium, the putrescine content rapidly increased and reached a peak, then fell gradually. Applying exogenous putrescine raised the embryogenesis frequency and endogenous putrescine level. It indicated that increase in putrescine content at early stage of differentiation promoted embryogenesis. With the development of somatic embryo, spermidine content reached its the highest level at globular embryo stage, spermine content rose and reached a peak at a later stage of globular embryo development. Furthermore, changes of the putrescine, spermidine and spermine contents during somatic embryogenesis were similar in Valencia calli which had different ploidy levels, but their contents decreased following the increasing of ploidy level. Changes in arginine decarboxylase activity were positively correlated to the polyamine levels, which suggest that the later is a key factor in regulating the polyamine levels during somatic embryogenesis in citrus plants.

Immunosensor for rapid detection of gibberellin acid in the rice grain.

A rapid, selective, sensitive, accurate, and inexpensive immunosensor for gibberellin acid detection was designed by coupling immunoassay with the square wave anodic stripping voltammetry (SWASV) technique involving copper ion labeled antigen in the competitive immunoreaction. The response signal expressed as the percentage of current reduction CR % (y) is linearly related to the concentration of GA (x) in the 1 microg/mL to approximately 150 microg/mL range with a regression equation of the form y = 0.44x + 15.59 and a correlation coefficient of 0.99. The results of the immunosensor assay were compared with those obtained by HPLC and ELISA, which show a satisfactory agreement. The immunosensor was used to determine the GA content in the hybrid rice grain samples taken in the growing period.

Amperometric immunosensor based on polypyrrole/poly(m-pheylenediamine) multilayer on glassy carbon electrode for cytokinin N6-(Delta2-isopentenyl) adenosine assay.

A novel immunosensor based on a multilayer-coated glassy carbon electrode was designed to determine isopentenyl adenosine (iPA) in plants. The multilayer consists of polypyrrole and poly(m-phenylenediamine) with K4Fe(CN)6 and horseradish peroxidase (HRP) entrapped during electropolymerization. The ferrocyanide doped in polypyrrole functions as the mediator. The glucose oxidase bound on the immunosensor by the competitive immunoreaction involving iPA catalyzed the oxidation of the added glucose with the formation of H2O2, which is in turn reduced in the presence of HRP entrapped in poly(m-phenylenediamine). The current of the oxidized production of ferrocyanide reduced at -50 mV is inversely proportional to the concentration of iPA in the competitive immunoreaction. This immunosensor is able to be used about 40 times; after that its surface can be regenerated for a new immunosensor assembly by washing with 0.1M citrate-phosphate buffer (pH 4.6). The percentage of current response reduction (CR%) (y) is linearly related to the logarithm of the concentration of iPA (x) in the 5-300 microg/ml range, with a regression equation of the form y = 42.13x - 27.79 and a correlation coefficient of 0.9861. Five hybrid rice grain samples were analyzed with results in satisfactory agreement to those obtained by high-performance liquid chromatography.

Amperometric biosensor with HRP immobilized on a sandwiched nano-Au / polymerized m-phenylenediamine film and ferrocene mediator.

An amperometric biosensor has been developed for the determination of H(2)O(2) in plant samples. Horseradish peroxidase (HRP) is immobilized on a sandwiched nano-Au particle / m-phenylenediamine polymer film by glutaraldehyde cross-linking. The film is formulated on the carbon paste electrode (CPE) blended with ferrocene as an electron transfer mediator. On the low concentration range, the current response is related to the H(2)O(2) concentration linearly from 0 to 8x10(-6) M with a detection limit of 1.3x10(-7) M. On a wider concentration range of 8x10(-6) to 1.4x10(-4) M, the reciprocal of current response is linearly related to the reciprocal of H(2)O(2) concentration. The apparent Michaelis-Menten constant (K(m)(app)) was calculated to be 0.0334 mM. The sensor has been tested by determining H(2)O(2) concentration in plant leaf samples.

A novel piezoelectric biosensor for the detection of phytohormone beta-indole acetic acid.

A novel piezoelectric immunosensor has been developed for the determination of beta-indole acetic acid (IAA) in dilute solutions. The detection is based on competitive immunoreaction between a hapten (IAA) and an antigen (IAA-BSA, hapten-protein conjugation) bound to an anti-IAA antibody, immobilized on a quartz crystal microbalance (QCM). The frequency change (y) of the sensor caused by antigen is linearly related to the logarithm of the concentration of IAA (x) in the range of 0.5 ng/ml - 5 microg/ml with a regression equation of the form y = -23x + 151 (r = 0.9937).