TMK-based cell-surface auxin signalling activates cell-wall acidification.

The phytohormone auxin controls many processes in plants, at least in part through its regulation of cell expansion1. The acid growth hypothesis has been proposed to explain auxin-stimulated cell expansion for five decades, but the mechanism that underlies auxin-induced cell-wall acidification is poorly characterized. Auxin induces the phosphorylation and activation of the plasma membrane H+-ATPase that pumps protons into the apoplast2, yet how auxin activates its phosphorylation remains unclear. Here we show that the transmembrane kinase (TMK) auxin-signalling proteins interact with plasma membrane H+-ATPases, inducing their phosphorylation, and thereby promoting cell-wall acidification and hypocotyl cell elongation in Arabidopsis. Auxin induced interactions between TMKs and H+-ATPases in the plasma membrane within seconds, as well as TMK-dependent phosphorylation of the penultimate threonine residue on the H+-ATPases. Our genetic, biochemical and molecular evidence demonstrates that TMKs directly phosphorylate plasma membrane H+-ATPase and are required for auxin-induced H+-ATPase activation, apoplastic acidification and cell expansion. Thus, our findings reveal a crucial connection between auxin and plasma membrane H+-ATPase activation in regulating apoplastic pH changes and cell expansion through TMK-based cell surface auxin signalling.

A mutation in CsHY2 encoding a phytochromobilin (PΦB) synthase leads to an elongated hypocotyl 1(elh1) phenotype in cucumber (Cucumis sativus L.).

KEY MESSAGE: The elongated hypocotyl1 (elh1) mutant in cucumber is due to a mutation in CsHY2, which is a homolog of the Arabidopsis HY2 encoding the phytochromobilin (PΦB) synthase for phytochrome biosynthesis Hypocotyl length is a critical determinant in establishing high quality seedlings for successful cucumber production, but knowledge on the molecular regulation of hypocotyl growth in cucumber is very limited. Here, we reported identification and characterization of a cucumber elongated hypocotyl 1 (elh1) mutant. We found that the longer hypocotyl in elh1 was due to longitudinal growth of hypocotyl cells. With fine mapping, the elh1 locus was delimited to a 20.9-kb region containing three annotated genes; only one polymorphism was identified in this region between two parental lines, which was a non-synonymous SNP (G28153633A) in the third exon of CsHY2 (CsGy1G030000) that encodes a phytochromobilin (PΦB) synthase. Uniqueness of the mutant allele at CsHY2 was verified in natural cucumber populations. Ectopic expression of CsHY2 in Arabidopsis hy2-1 long-hypocotyl mutant led to reduced hypocotyl length. The PΦB protein was targeted to the chloroplast. The expression levels of CsHY2 and five phytochrome genes CsPHYA1, CsPHYA2, CsPHYB, CsPHYC and CsPHYE were all significantly down-regulated while several cell elongation related genes were up-regulated in elh1 mutant compared to wild-type cucumber, which are correlated with dynamic hypocotyl elongation in the mutant. RNA-seq analysis in the WT and mutant revealed differentially expressed genes involved in porphyrin and chlorophyll metabolisms, cell elongation and plant hormone signal transduction pathways. This is the first report to characterize and clone the CsHY2 gene in cucumber. This work reveals the important of CsHY2 in regulating hypocotyl length and extends our understanding of the roles of CsHY2 in cucumber.

Mapping and identification of CsSh5.1, a gene encoding a xyloglucan galactosyltransferase required for hypocotyl elongation in cucumber (Cucumis sativus L.).

KEY MESSAGE: CsSh5.1, which controls hypocotyl elongation under high temperature conditions in cucumber, was mapped to a 57.1 kb region on chromosome 5 containing a candidate gene encoding a xyloglucan galactosyltransferase. Hypocotyl growth is a vital process in seedling establishment. Hypocotyl elongation after germination relies more on longitudinal cell elongation than cell division. Cell elongation is largely determined by the extensibility of the cell wall. Here, we identified a spontaneous mutant in cucumber (Cucumis sativus L.), sh5.1, which exhibits a temperature-insensitive short hypocotyl phenotype. Genetic analysis showed that the phenotype of sh5.1 was controlled by a recessive nuclear gene. CsSh5.1 was mapped to a 57.1 kb interval on chromosome 5, containing eight predicted genes. Sequencing analysis revealed that the Csa5G171710 is the candidate gene of CsSh5.1, which was further confirmed via co-segregation analysis and genomic DNA sequencing in natural cucumber variations. The result indicated that hypocotyl elongation might be controlled by this gene. CsSh5.1 encodes a xyloglucan galactosyltransferase that specifically adds galactose to xyloglucan and forms galactosylated xyloglucans, which determine the strength and extensibility of the cell walls. CsSh5.1 expression in wild-type (WT) hypocotyl was significantly higher than that in sh5.1 hypocotyl under high temperature, suggesting its important role in hypocotyl cell elongation under high temperature. The identification of CsSh5.1 is helpful for elucidating the function of xyloglucan galactosyltransferase in cell wall expansion and understanding the mechanism of hypocotyl elongation in cucumber.

The apple RING-H2 protein MdCIP8 regulates anthocyanin accumulation and hypocotyl elongation by interacting with MdCOP1.

COP1, an important RING ubiquitin ligase E3, is a molecular switch for light regulation in plant development. As an interacting protein of COP1, CIP8 contains a RING-H2 domain, but its biological function is unclear. Here, the apple MdCIP8 was identified based on its homology with AtCIP8 in Arabidopsis. MdCIP8 was constitutively expressed at different levels in various apple tissues, and the expression level of MdCIP8 was not affected by light and dark conditions. MdCIP8 reversed the short hypocotyl phenotype of the cip8 mutant under light conditions. Furthermore, the yeast two-hybrid experiment showed that MdCIP8 interacted with the RING domain of MdCOP1 through its RING-H2 domain. MdCIP8-OX/cop1-4 exhibited the phenotype of the cop1-4 mutant, indicating that CIP8 acts upstream of COP1. In addition, an apple transient injection experiment showed that MdCIP8 inhibited anthocyanin accumulation in an MdCOP1-dependent pathway. Overall, our findings reveal that CIP8 plays an inhibitory role in the light-regulation responses of plants.

The Arabidopsis RLCK VI_A2 Kinase Controls Seedling and Plant Growth in Parallel with Gibberellin.

The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation.

Salicylic acid regulates adventitious root formation via competitive inhibition of the auxin conjugation enzyme CsGH3.5 in cucumber hypocotyls.

MAIN CONCLUSION: Exogenous SA treatment at appropriate concentrations promotes adventitious root formation in cucumber hypocotyls, via competitive inhibiting the IAA-Asp synthetase activity of CsGH3.5, and increasing the local free IAA level. Adventitious root formation is critical for the cutting propagation of horticultural plants. Indole-3-acetic acid (IAA) has been shown to play a central role in regulating this process, while for salicylic acid (SA), its exact effects and regulatory mechanism have not been elucidated. In this study, we showed that exogenous SA treatment at the concentrations of both 50 and 100 µM promoted adventitious root formation at the base of the hypocotyl of cucumber seedlings. At these concentrations, SA could induce the expression of CYCLIN and Cyclin-dependent Kinase (CDK) genes during adventitious rooting. IAA was shown to be involved in SA-induced adventitious root formation in cucumber hypocotyls. Exposure to exogenous SA led to a slight increase in the free IAA content, and pre-treatment with the auxin transport inhibitor 1-naphthylphthalamic acid (NPA) almost completely abolished the inducible effects of SA on adventitious root number. SA-induced IAA accumulation was also associated with the enhanced expression of Gretchen Hagen3.5 (CsGH3.5). The in vitro enzymatic assay indicated that CsGH3.5 has both IAA- and SA-amido synthetase activity and prefers aspartate (Asp) as the amino acid conjugate. The Asp concentration dictated the functional activity of CsGH3.5 on IAA. Both affinity and catalytic efficiency (Kcat/Km) increased when the Asp concentration increased from 0.3 to 1 mM. In contrast, CsGH3.5 showed equal catalytic efficiency for SA at low and high Asp concentrations. Furthermore, SA functioned as a competitive inhibitor of the IAA-Asp synthetase activity of CsGH3.5. During adventitious formation, SA application indeed repressed the IAA-Asp levels in the rooting zone. These data show that SA plays an inducible role in adventitious root formation in cucumber through competitive inhibition of the auxin conjugation enzyme CsGH3.5. SA reduces the IAA conjugate levels, thereby increasing the local free IAA level and ultimately enhancing adventitious root formation.

Toward alternative sources of milk coagulants for cheese manufacturing: establishment of hairy roots culture and protease characterization from Cynara cardunculus L.

KEY MESSAGE: Extracts from hairy root cultures of Cynara cardunculus L. contain proteases and show milk-clotting activity. Cynara cardunculus L. or cardoon is often used as rennet in traditional cheese manufacturing, due to the presence of specific proteases in the flower. However, the flower extracts are variable depending on the provenance and quality of the flowers as well as high genetic variability among cardoon populations, and this affects the quality of the final product. In search for alternative sources of milk-clotting enzymes, hairy root cultures from cardoon were obtained and characterized regarding their protease content and proteolytic activity toward milk proteins. Aspartic, serine and cysteine proteases were identified in hairy roots by mass spectrometry analysis and an azocasein assay combined with specific inhibitors. RT-PCR analysis revealed the expression of cardosin A and D, and immunoblotting analysis suggested the presence of cardosin A or cardosin A-like enzyme in its mature form, supporting this system as an alternative source of cardosins. Hairy root protein extracts showed activity over caseins, supporting its use as milk coagulant, which was further tested by milk-clotting assays. This is also the first report on the establishment of hairy root cultures from cardoon, which paves the way for future work on controlled platforms for production of valuable metabolites which are known to be present in this species.

Sumoylation of BRI1-EMS-SUPPRESSOR 1 (BES1) by the SUMO E3 Ligase SIZ1 Negatively Regulates Brassinosteroids Signaling in Arabidopsis thaliana.

Brassinosteroids (BRs), a group of plant steroid hormones, participate in the regulation of plant growth and developmental processes. BR functions through the BES1/BZR1 family of transcription factors, however, the regulation of the BES1 activity by post-translational modifications remains largely unknown. Here, we present evidence that the SUMO E3 ligase SIZ1 negatively regulates BR signaling pathway. T-DNA insertion mutant siz1-2 shows BL (Brassinolide, the most active BR) hypersensitivity and BRZ (Brassinazole, a BR biosynthesis inhibitor) insensitivity during hypocotyl elongation. In addition, expression of BES1-dependent BR-response genes is hyper-regulated in siz1-2 seedlings. The siz1-2bes1-D double mutant exhibits longer hypocotyl than bes1-D. Moreover, SIZ1 physically interacts with BES1 in vivo and in vitro and mediates the sumoylation of BES1. A K302R substitution in BES1 blocks its sumoylation mediated by SIZ1 in plants, indicating that K302 is the principal site for SUMO conjugation. Consistently, we find that sumoylation inhibits BES1 protein stability and activity. Taken together, our data show that the sumoylation of BES1 via SIZ1 negatively regulates the BR signaling pathway.

Nitric oxide induced modulations in adventitious root growth, lignin content and lignin synthesizing enzymes in the hypocotyls of Vigna radiata.

The present study evaluated the role of nitric oxide (NO) in mediating adventitious root (AR) growth, lignification and related enzymatic changes in the hypocotyls of Vigna radiata. To meet the objectives, the changes in AR growth, lignin content, and the activities of enzymes-peroxidases, polyphenol oxidases, and phenylalanine ammonia lyases- with NO donor and its scavenger were monitored. Hypocotyls were cultivated in aqueous solution supplemented with different concentrations of SNP (sodium nitroprusside, NO donor compound) and its scavenging compound (2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; cPTIO). Specifically, at low concentrations, SNP induced AR growth, increased the total lignin content and altered the activities of related oxidoreductases- peroxidases, polyphenol oxidases and phenylalanine ammonia lyases- which are involved in lignin biosynthesis pathway. At higher concentrations, a decline in AR growth and lignification was noticed. We analysed the function of NO in AR formation by depleting the endogenous NO using scavenging compound cPTIO. Hypocotyls grown in a medium supplemented with scavenger cPTIO exhibited significant decline in AR growth and the activities of lignin synthesizing enzymes. Application of NO scavenger showed that stimulatory properties on root lignification may be owing to NO itself. In addition, changes in AR growth were significantly correlated with these modified biochemical activities. Our analysis revealed that NO supplementation induces prominent alterations in lignin level during AR formation and this might be due to an alteration in the activity of lignin biosynthetic enzymes, which further affected the polymerization of monolignols and AR growth.

Involvement of PP6-type protein phosphatase in hypocotyl phototropism in Arabidopsis seedlings.

Recently, we reported that the D6 protein kinase subfamily, which belongs to the AGCVIII kinase family, is a critical component of hypocotyl phototropism in Arabidopsis seedlings. Furthermore, we demonstrated that AGC1-12, which is also a member of the AGCVIII kinase family, is involved in both the pulse-induced first positive phototropism and gravitropism in Arabidopsis hypocotyls. Those results indicated that phosphorylation control is an important mechanism in phototropic signaling. As phosphorylation regulation is controlled by both kinases and phosphatases, we investigated the roles of phosphatases in hypocotyl phototropism. Our physiological analysis, which was performed using Arabidopsis mutants, indicated that the flower-specific, phytochrome-associated protein phosphatase family, which functions as a catalytic subunit of protein phosphatase 6 (PP6), is involved in both the pulse-induced first positive phototropism and the time-dependent second positive phototropism, although it is not necessary for the continuous-light-induced second positive phototropism. These results suggest that not only kinases, but also phosphatases play critical roles in hypocotyl phototropism to control phosphorylation status and that PP6-type protein phosphatases may act antagonistically with AGCVIII protein kinases on the same targets, such as PIN-formed proteins.

Roles of AGCVIII Kinases in the Hypocotyl Phototropism of Arabidopsis Seedlings.

Regulation of protein function by phosphorylation and dephosphorylation is an important mechanism in many cellular events. The phototropin blue-light photoreceptors, plant-specific AGCVIII kinases, are essential for phototropic responses. Members of the D6 PROTEIN KINASE (D6PK) family, representing a subfamily of the AGCVIII kinases, also contribute to phototropic responses, suggesting that possibly further AGCVIII kinases may potentially control phototropism. The present study investigates the functional roles of Arabidopsis (Arabidopsis thaliana) AGCVIII kinases in hypocotyl phototropism. We demonstrate that D6PK family kinases are not only required for the second but also for the first positive phototropism. In addition, we find that a previously uncharacterized AGCVIII protein, AGC1-12, is involved in the first positive phototropism and gravitropism. AGC1-12 phosphorylates serine residues in the cytoplasmic loop of PIN-FORMED 1 (PIN1) and shares phosphosite preferences with D6PK. Our work strongly suggests that the D6PK family and AGC1-12 are critical components for both hypocotyl phototropism and gravitropism, and that these kinases control tropic responses mainly through regulation of PIN-mediated auxin transport by protein phosphorylation.

Implications of the galactosidase activity of yieldin in the regulatory mechanism of yield threshold that is fundamental to cell wall extension.

To understand the action mechanism of yieldin (YLD) on the regulation of the yield threshold (Y), one of the critical parameters of cell wall extension, YLD was extracted from the cell walls of cowpea (Vigna unguiculata L.) hypocotyls and the hemagglutinin activity (HA) as well as the glycosidase activity of the protein was measured. Sedimentation assays using trypsinated rabbit erythrocytes showed that YLD possessed HA at pH 7. The digestion assays using 4-nitrophenyl (pNP) glycopyranosides as artificial substrates showed that YLD liberated galactose residues from pNP alpha-d-galactopyranoside mainly at pH 4.0, i.e. the pH level where Y was decreased at most. These results show that YLD is a bifunctional protein that switches between the HA and the galactosidase activities depending on the surrounding pH. Since D-galactose at concentration of 0.03 g l-1 perfectly inhibited the HA, YLD was suggested to associate with galactose residues. However, the galactose application ten times concentrated was necessary to inhibit both the galactosidase activity of YLD and the acid-induced shift of Y regulated by YLD. In addition, the specific inhibitor of alpha-d-galactosidase (deoxygalactonojirimycin) inhibited both the galactosidase activity of YLD and the shift of Y at the same concentration, but not the HA. On the basis of these results, it is suggested the galactosidase activity of YLD plays a central role in the mechanism of Y-regulation at acidic pH.

A mutation of casein kinase 2 α4 subunit affects multiple developmental processes in Arabidopsis.

KEY MESSAGE: Arabidopsis CK2 α4 subunit regulates the primary root and hypocotyl elongation, lateral root formation, cotyledon expansion, rosette leaf initiation and growth, flowering, and anthocyanin biosynthesis. Casein kinase 2 (CK2) is a conserved tetrameric kinase composed of two α and two ß subunits. The inhibition of CK2 activity usually results in severe developmental deficiency. Four genes (CKA1-CKA4) encode CK2 α subunit in Arabidopsis. Single mutations of CKA1, CKA2, and CKA3 do not affect the normal growth of Arabidopsis, while the cka1 cka2 cka3 triple mutants are defective in cotyledon and hypocotyl growth, lateral root development, and flowering. The inhibition of CKA4 expression in cka1 cka2 cka3 background further reduces the number of lateral roots and delays the flowering time. Here, we report the characterization of a novel knockout mutant of CKA4, which exhibits various developmental defects including reduced primary root and hypocotyl elongation, increased lateral root density, delayed cotyledon expansion, retarded rosette leaf initiation and growth, and late flowering. The examination of the cellular basis for abnormal root development of this mutant revealed reduced root meristem cells with enhanced RETINOBLASTOMA-RELATED (RBR) expression that promotes cell differentiation in root meristem. Moreover, this cka4-2 mutant accumulates higher anthocyanin in the aerial part and shows an increased expression of anthocyanin biosynthetic genes, suggesting a novel role of CK2 in modulating anthocyanin biosynthesis. In addition, the complementation test using primary root elongation assay as a sample confirms that the changed phenotypes of this cka4-2 mutant are due to the lack of CKA4. Taken together, this study reveals an essential role of CK2 α4 subunit in multiple developmental processes in Arabidopsis.

Identification of a Highly Specific Isoflavone 7-O-glucosyltransferase in the soybean (Glycine max (L.) Merr.).

Isoflavone conjugates [7-O-ß-D-glucosides and 7-O-(6″-malonyl-ß-D-glucosides) of daidzein and genistein] accumulate in soybean roots and serve as the stored precursors of isoflavones (aglycons), which play very important roles in the rhizobia-mediated nodulation of this plant. Thus far, the isoflavone 7-O-glucosyltransferase (GmIF7GT or GmUGT1) has been biochemically characterized and is believed to be involved in isoflavone conjugate biosynthesis. The soybean genome encodes many other glycosyltransferase homologs (GmUGTs) that are related to GmUGT1; however, their catalytic properties, substrate specificities, and role(s) in isoflavone conjugation are unknown. In this study, nine different GmUGT1-related GmUGT cDNAs were isolated; six of these cDNAs belonged to two distinct phylogenetic subgroups (A and B), and these six were functionally characterized. The results showed that GmUGT4, a representative of subgroup A, encoded a UGT that was highly specific for isoflavones showing kcat and kcat/Km values for daidzein of 5.89 ± 0.65 s(-1) and 2.91 × 10(5) s(-1)M(-1), respectively. Moreover, GmUGT4 was expressed in the roots (mainly in lateral roots) of the 7-day-old seedlings and seeds, both of which contained abundant amounts of isoflavone conjugates. By contrast, GmUGT1 and GmUGT7, which were subgroup B members, encoded enzymes with broad glucosyl-acceptor specificities and were mainly expressed in the aerial portions (cotyledons and hypocotyls) of the seedlings. In the present study, we proposed that the role of isoflavone glucosylation in a soybean plant is assigned to different GmUGT members in an organ/tissue-dependent manner. We also established the functional importance of GmUGT4 in the biosynthesis of isoflavone conjugates in lateral roots that make a major contribution to overall N2 fixation.

Ribonuclease J is required for chloroplast and embryo development in Arabidopsis.

Chloroplasts perform many essential metabolic functions and their proper development is critically important in embryogenesis. However, little is known about how chloroplasts function in embryogenesis and more relevant components need to be characterized. In this study, we show that Arabidopsis Ribonuclease J (RNase J) is required for chloroplast and embryo development. Mutation of AtRNJ led to albino ovules containing aborted embryos; the morphological development of rnj embryos was disturbed after the globular stage. Observation of ultrastructures indicated that these aborted embryos may result from impaired chloroplast development. Furthermore, by analyzing the molecular markers of cell fate decisions (STM, FIL, ML1, SCR, and WOX5) in rnj embryos, we found that this impairment of chloroplast development may lead to aberrant embryo patterning along the apical-basal axis, indicating that AtRNJ is important in initiating and maintaining the organization of shoot apical meristems (SAMs), cotyledons, and hypocotyls. Moreover, the transport and response of auxin in rnj embryos was found to be disrupted, suggesting that AtRNJ may be involved in auxin-mediated pathways during embryogenesis. Therefore, we speculate that RNJ plays a vital role in embryo morphogenesis and apical-basal pattern formation by regulating chloroplast development.

Promotion of testa rupture during garden cress germination involves seed compartment-specific expression and activity of pectin methylesterases.

Pectin methylesterase (PME) controls the methylesterification status of pectins and thereby determines the biophysical properties of plant cell walls, which are important for tissue growth and weakening processes. We demonstrate here that tissue-specific and spatiotemporal alterations in cell wall pectin methylesterification occur during the germination of garden cress (Lepidium sativum). These cell wall changes are associated with characteristic expression patterns of PME genes and resultant enzyme activities in the key seed compartments CAP (micropylar endosperm) and RAD (radicle plus lower hypocotyl). Transcriptome and quantitative real-time reverse transcription-polymerase chain reaction analysis as well as PME enzyme activity measurements of separated seed compartments, including CAP and RAD, revealed distinct phases during germination. These were associated with hormonal and compartment-specific regulation of PME group 1, PME group 2, and PME inhibitor transcript expression and total PME activity. The regulatory patterns indicated a role for PME activity in testa rupture (TR). Consistent with a role for cell wall pectin methylesterification in TR, treatment of seeds with PME resulted in enhanced testa permeability and promoted TR. Mathematical modeling of transcript expression changes in germinating garden cress and Arabidopsis (Arabidopsis thaliana) seeds suggested that group 2 PMEs make a major contribution to the overall PME activity rather than acting as PME inhibitors. It is concluded that regulated changes in the degree of pectin methylesterification through CAP- and RAD-specific PME and PME inhibitor expression play a crucial role during Brassicaceae seed germination.

Glycosyltransferase-like protein ABI8/ELD1/KOB1 promotes Arabidopsis hypocotyl elongation through regulating cellulose biosynthesis.

Seedling de-etiolation (photomorphogenesis) is an important light-regulated developmental process in plants. Here, we showed that disruption of the gene encoding a glycosyltransferase-like protein, ABA INSENSITIVE 8 (ABI8)/ELONGATION EFFECTIVE 1 (ELD1)/KOBITO1 (KOB1), caused short-hypocotyl elongation under all light conditions examined and even in darkness. We found that the ABI8 transcript level was down-regulated by light in a phytochrome A-dependent manner. Furthermore, light destabilized ABI8 protein via the 26S proteasome degradation pathway. We showed that ABI8 promoted the expression of genes involved in cell elongation and cellulose synthesis. Consistently, the cellulose content was reduced in the abi8 mutants and application of 2, 6-dichlorobenzonitrile (an inhibitor of cellulose biosynthesis) mimicked the abi8 mutant phenotype. Moreover, we found that phytochrome and cryptochrome photoreceptors negatively, whereas CONSTITUTIVE PHOTOMORPHOGENIC 1 positively, regulated cellulose synthesis. We also showed that ELONGATED HYPOCOTYL 5 directly bound to the promoters of ABI8 and several cellulose synthesis genes and repressed their expression in light conditions. Taken together, our study reveals that ABI8 functions as a negative factor in light inhibition of hypocotyl elongation through modulating cellulose biosynthesis.

A comparative proteomic analysis of responses to high temperature stress in hypocotyl of Canola (Brassica napus L.).

High temperature stress, especially on the early season of plant growth stages, is an agricultural problem in many areas in the world. A temporary or continually high temperature leads to a set of morphological, biochemical and physiological changes in plants, which consequently reduces the plant growth and development and finally may cause a severe reduction in economic yield. The main goal of this study was to assess plant response to high temperature stress (HTS) in early seedling of canola. This study is the first experiment on the effect of heat stress on proteome of canola. In the present research, a proteomics approach was used to evaluate the effects of high temperature stress, including 45 °C day/34 °C night for 2, 6 and 12 hour, on early seedling stage (2-day old) of canola. Proteins were isolated from hypocotyl and separated by two-dimensional polyacrylamide gel electrophoresis. Out of 381 protein spots, 28 and 34 proteins were significantly down- and up-regulated, respectively. The trend of mRNA expression for sucrose binding protein, a scorbate peroxidase and triosephosphateisomerase, was in accordance with their trend at translation level. Results of this study suggest that the up-regulation of proteins involved in cellular traffic, energy and metabolism, and down-regulation of some proteins involved in disease and defense, protein synthesis and signal transduction could be the main reason of physiological and morphological responses to high temperature stress. The observed increases in the level of ascorbate peroxidase protein and mRNA expression in canola hypocotyl in response to HTS suggests that ascorbate peroxidase is a short term high temperature stress response protein and is thus a candidate for gene modification strategies aimed at producing high temperature canola varieties. These results also suggest that the up regulation of protein involved in energy and metabolism in response to the heat stress can use most of nutritive reserves in seedling of canola and might explain the reduced growth of canola in heat stress conditions.

Indaziflam herbicidal action: a potent cellulose biosynthesis inhibitor.

Cellulose biosynthesis is a common feature of land plants. Therefore, cellulose biosynthesis inhibitors (CBIs) have a potentially broad-acting herbicidal mode of action and are also useful tools in decoding fundamental aspects of cellulose biosynthesis. Here, we characterize the herbicide indaziflam as a CBI and provide insight into its inhibitory mechanism. Indaziflam-treated seedlings exhibited the CBI-like symptomologies of radial swelling and ectopic lignification. Furthermore, indaziflam inhibited the production of cellulose within <1 h of treatment and in a dose-dependent manner. Unlike the CBI isoxaben, indaziflam had strong CBI activity in both a monocotylonous plant (Poa annua) and a dicotyledonous plant (Arabidopsis [Arabidopsis thaliana]). Arabidopsis mutants resistant to known CBIs isoxaben or quinoxyphen were not cross resistant to indaziflam, suggesting a different molecular target for indaziflam. To explore this further, we monitored the distribution and mobility of fluorescently labeled CELLULOSE SYNTHASE A (CESA) proteins in living cells of Arabidopsis during indaziflam exposure. Indaziflam caused a reduction in the velocity of YELLOW FLUORESCENT PROTEIN:CESA6 particles at the plasma membrane focal plane compared with controls. Microtubule morphology and motility were not altered after indaziflam treatment. In the hypocotyl expansion zone, indaziflam caused an atypical increase in the density of plasma membrane-localized CESA particles. Interestingly, this was accompanied by a cellulose synthase interacting1-independent reduction in the normal coincidence rate between microtubules and CESA particles. As a CBI, for which there is little evidence of evolved weed resistance, indaziflam represents an important addition to the action mechanisms available for weed management.

Gibberellin overproduction promotes sucrose synthase expression and secondary cell wall deposition in cotton fibers.

Bioactive gibberellins (GAs) comprise an important class of natural plant growth regulators and play essential roles in cotton fiber development. To date, the molecular base of GAs' functions in fiber development is largely unclear. To address this question, the endogenous bioactive GA levels in cotton developing fibers were elevated by specifically up-regulating GA 20-oxidase and suppressing GA 2-oxidase via transgenic methods. Higher GA levels in transgenic cotton fibers significantly increased micronaire values, 1000-fiber weight, cell wall thickness and cellulose contents of mature fibers. Quantitative RT-PCR and biochemical analysis revealed that the transcription of sucrose synthase gene GhSusA1 and sucrose synthase activities were significantly enhanced in GA overproducing transgenic fibers, compared to the wild-type cotton. In addition, exogenous application of bioactive GA could promote GhSusA1 expression in cultured fibers, as well as in cotton hypocotyls. Our results suggested that bioactive GAs promoted secondary cell wall deposition in cotton fibers by enhancing sucrose synthase expression.