OsPEX1, an extensin-like protein, negatively regulates root growth in a gibberellin-mediated manner in rice.

Leucine-rich repeat extensins (LRXs) are required for plant growth and development through affecting cell growth and cell wall formation. LRX gene family can be classified into two categories: predominantly vegetative-expressed LRX and reproductive-expressed PEX. In contrast to the tissue specificity of Arabidopsis PEX genes in reproductive organs, rice OsPEX1 is also highly expressed in roots in addition to reproductive tissue. However, whether and how OsPEX1 affects root growth is unclear. Here, we found that overexpression of OsPEX1 retarded root growth by reducing cell elongation likely caused by an increase of lignin deposition, whereas knockdown of OsPEX1 had an opposite effect on root growth, indicating that OsPEX1 negatively regulated root growth in rice. Further investigation uncovered the existence of a feedback loop between OsPEX1 expression level and GA biosynthesis for proper root growth. This was supported by the facts that exogenous GA3 application downregulated transcript levels of OsPEX1 and lignin-related genes and rescued the root developmental defects of the OsPEX1 overexpression mutant, whereas OsPEX1 overexpression reduced GA level and the expression of GA biosynthesis genes. Moreover, OsPEX1 and GA showed antagonistic action on the lignin biosynthesis in root. OsPEX1 overexpression upregulated transcript levels of lignin-related genes, whereas exogenous GA3 application downregulated their expression. Taken together, this study reveals a possible molecular pathway of OsPEX1mediated regulation of root growth through coordinate modulation of lignin deposition via a negative feedback regulation between OsPEX1 expression and GA biosynthesis.

Overexpression of Pennisetum purpureum CCoAOMT Contributes to Lignin Deposition and Drought Tolerance by Promoting the Accumulation of Flavonoids in Transgenic Tobacco.

Elephant grass (Pennisetum purpureum) is a fast-growing and low-nutrient demand plant that is widely used as a forage grass and potential energy crop in tropical and subtropical regions of Asia, Africa, and the United States. Transgenic tobacco with the PpCCoAOMT gene from Pennisetum purpureum produces high lignin content that is associated with drought tolerance in relation to lower accumulation of reactive oxygen species (ROS), along with higher antioxidant enzyme activities and osmotic adjustment. In this study, transgenic tobacco plants revealed no obvious cost to plant growth when expressing the PpCCoAOMT gene. Metabolomic studies demonstrated that tobacco plants tolerant to drought stress accumulated flavonoids under normal and drought conditions, which likely explains the observed tolerance phenotype in wild-type tobacco. Our results suggest that plants overexpressing PpCCoAOMT were better able to cope with water deficit than were wild-type controls; metabolic flux was redirected within primary and specialized metabolism to induce metabolites related to defense to drought stress. These results could help to develop drought-resistant plants for agriculture in the future.

Overexpression of SgGH3.1 from Fine-Stem Stylo (Stylosanthes guianensis var. intermedia) Enhances Chilling and Cold Tolerance in Arabidopsis thaliana.

Stylosanthes (stylo) species are commercially significant tropical and subtropical forage and pasture legumes that are vulnerable to chilling and frost. However, little is known about the molecular mechanisms behind stylos' responses to low temperature stress. Gretchen-Hagen 3 (GH3) proteins have been extensively investigated in many plant species for their roles in auxin homeostasis and abiotic stress responses, but none have been reported in stylos. SgGH3.1, a cold-responsive gene identified in a whole transcriptome profiling study of fine-stem stylo (S. guianensis var. intermedia) was further investigated for its involvement in cold stress tolerance. SgGH3.1 shared a high percentage of identity with 14 leguminous GH3 proteins, ranging from 79% to 93%. Phylogenetic analysis classified SgGH3.1 into Group Ⅱ of GH3 family, which have been proven to involve with auxins conjugation. Expression profiling revealed that SgGH3.1 responded rapidly to cold stress in stylo leaves. Overexpression of SgGH3.1 in Arabidopsis thaliana altered sensitivity to exogenous IAA, up-regulated transcription of AtCBF1-3 genes, activated physiological responses against cold stress, and enhanced chilling and cold tolerances. This is the first report of a GH3 gene in stylos, which not only validated its function in IAA homeostasis and cold responses, but also gave insight into breeding of cold-tolerant stylos.

Functional characterization of PETIOLULE-LIKE PULVINUS (PLP) gene in abscission zone development in Medicago truncatula and its application to genetic improvement of alfalfa.

Alfalfa (Medicago sativa L.) is one of the most important forage crops throughout the world. Maximizing leaf retention during the haymaking process is critical for achieving superior hay quality and maintaining biomass yield. Leaf abscission process affects leaf retention. Previous studies have largely focused on the molecular mechanisms of floral organ, pedicel and seed abscission but scarcely touched on leaf and petiole abscission. This study focuses on leaf and petiole abscission in the model legume Medicago truncatula and its closely related commercial species alfalfa. By analysing the petiolule-like pulvinus (plp) mutant in M. truncatula at phenotypic level (breakstrength and shaking assays), microscopic level (scanning electron microscopy and cross-sectional analyses) and molecular level (expression level and expression pattern analyses), we discovered that the loss of function of PLP leads to an absence of abscission zone (AZ) formation and PLP plays an important role in leaflet and petiole AZ differentiation. Microarray analysis indicated that PLP affects abscission process through modulating genes involved in hormonal homeostasis, cell wall remodelling and degradation. Detailed analyses led us to propose a functional model of PLP in regulating leaflet and petiole abscission. Furthermore, we cloned the PLP gene (MsPLP) from alfalfa and produced RNAi transgenic alfalfa plants to down-regulate the endogenous MsPLP. Down-regulation of MsPLP results in altered pulvinus structure with increased leaflet breakstrength, thus offering a new approach to decrease leaf loss during alfalfa haymaking process.

SgRVE6, a LHY-CCA1-Like Transcription Factor From Fine-Stem Stylo, Upregulates NB-LRR Gene Expression and Enhances Cold Tolerance in Tobacco.

Stylosanthes species are economically important tropical and subtropical forage legumes which are generally vulnerable to chilling and frost. Fine-stem stylo (S. guianensis var. intermedia) has the most superior cold tolerance among all stylo species. A REVEILLE (RVE) gene, SgRVE6, was cloned from fine-stem stylo. Bioinformatic analysis suggests that SgRVE6 encodes a transcription factor of 292 amino acid residues, which belongs to the LATE ELONGATED HYPOCOTYL/CIRCADIAN CLOCK ASSOCIATED 1-LIKE (LCL) subgroup of RVE family and contains a SHAQKYF-class MYB domain and a LCL domain. SgRVE6 is universally expressed in root, stem and leaf tissues of fine-stem stylo and is rapidly up-regulated in all tested tissues under cold stress. Over-expressing SgRVE6 affects expression of 21 circadian clock genes, up-regulates expression of 6 nucleotide binding domain leucine-rich repeats (NB-LRR) encoding genes associated with tobacco cold tolerance, improves physiological responses to low temperature, and endows the transgenic tobaccos with higher tolerance to cold stress. This is the first time a study investigates the biological function of RVE6 in cold responses of plant species.

Vascular preferential activity of the Pennisetum purpureum cinnamyl alcohol dehydrogenase promoter in transgenic tobacco plants.

Little is known about the cross talk between the lignin biosynthesis gene promoters and the regulatory proteins that modulate molecular signaling and respond to various stresses. In this study, we characterized the promoter region of the lignin biosynthesis pathway cinnamyl alcohol dehydrogenase (CAD) gene in elephant grass, Pennisetum purpureum. Quantification of the transcript levels of the PpCAD promoter revealed it is preferentially expressed in vascular tissue, especially xylem. Histochemical and fluorometric assays confirmed the vascular-preferential expression of the PpCAD promoter, as the highest ß-glucuronidase (GUS) activity was found in the basal stem in transgenic tobacco plants expressing a 1154-bp PpCAD promoter-GUS fusion construct. Moreover, 5'-deleted PpCAD promoter analyses showed that the 1154-bp PpCAD promoter fragment had the highest transcriptional activity, whereas the 2054-bp fragment had multifarious inducible activity responding to gibberellin (GA), methyl jasmonate (MeJA), abscisic acid (ABA), and wounding. The regions from -248 to -243 bp and -1416 to -1411 bp contained W-box cis-elements, which were detected by electrophoretic mobility shift assay (EMSA). The binding effects of the GA-responsive elements (from -561 to -555 bp and -1077 to -1071 bp), MeJA-responsive element (from -1146 to -1142 bp), and the ABA-responsive cis-element (from -1879 to -1874 bp) were also validated by EMSA. Based on our results, we suggest that lignin deposition associated with PpCAD promoter activity adapts to the environment through molecular signaling involving GA, MeJA, and ABA.

Analysis of gene expression in Kalanchoe daigremontiana leaves during plantlet formation under drought stress

Background: Kalanchoe daigremontiana is an attractive model system for the study of the molecular mechanisms of somatic embryogenesis and organogenesis competence due to its formation of plantlets with adventitious roots on the leaf margins that are derived from somatic embryos. The suppression subtractive hybridization technique was used to investigate gene expression during asexual reproduction. Leaves from plants subjected to drought stress provided the source of ‘Tester’ DNA, and leaves from plants grown under normal conditions provided the ‘Driver’ DNA for subtractive hybridization. Results: A total of 481 high quality ESTs were generated, which clustered into 390 unigenes. Of these unigenes, 132 grouped into 12 functional categories, suggesting that asexual reproduction is a complicated process involving a large number of genes. The expression characteristics of selected genes from the SSH library were determined by real-time PCR and were classified into five groups, suggesting that gene expression patterns during asexual reproduction are complex. Up-regulation of S-adenosylhomocysteine hydrolase suggested that a decrease in cytokinin levels promotes the initiation of plantlet formation. Many other genes, such as inorganic pyrophosphatase and glutamate decarboxylase, play important roles in gene regulation during asexual reproduction. Conclusion: Our results provide a framework and unified platform on which future research on asexual reproduction in K. daigremontiana can be based. This represents the first genome-wide study of asexual reproduction in K. daigremontiana.