In recent years, peptide hormones have been recognized as important signal molecules in plants. Genetic characterization of such peptides is challenging since they are usually encoded by small genes. As a proof of concept, we used the wellcharacterized stem cell-restricting CLAVATA3 (CLV3) to develop an antagonistic peptide technology by transformations of wild-type Arabidopsis (Arabidopsis thaliana) with constructs carrying the full-length CLV3 with every residue in the peptidecoding region replaced, one at a time, by alanine. Analyses of transgenic plants allowed us to identify one line exhibiting a dominant-negative clv3-like phenotype, with enlarged shoot apical meristems and increased numbers of floral organs. We then performed second dimensional amino acid substitutions to replace the glycine residue individually with the other 18 possible proteinaceous amino acids. Examination of transgenic plants showed that a glycine-to-threonine substitution gave the strongest antagonistic effect in the wild type, in which over 70% of transgenic lines showed the clv3-like phenotype. Among these substitutions, a negative correlation was observed between the antagonistic effects in the wild type and the complementation efficiencies in clv3. We also demonstrated that such an antagonistic peptide technology is applicable to other CLV3/EMBRYO SURROUNDING REGION (CLE) genes, CLE8 and CLE22, as well as in vitro treatments. We believe this technology provides a powerful tool for functional dissection of widely occurring CLE genes in plants.

As a peptide hormone, CLV3 restricts the stem cell number in shoot apical meristem (SAM) by interacting with CLV1/CLV2/CRN/RPK2 receptor complexes. To elucidate how the function of the CLV3 peptide in SAM maintenance is established at the amino acid (AA) level, alanine substitutions were performed by introducing point mutations to individual residues in the peptide-coding region of CLV3 and its flanking sequences. Constructs carrying such substitutions, expressed under the control of CLV3 regulatory elements, were transformed to the clv3-2 null mutant to evaluate their efficiencies in complementing its defects in SAMs in vivo. These studies showed that aspartate-8, histidine-11, glycine-6, proline-4, arginine-1, and proline-9, arranged in an order of importance, were critical, while threonine-2, valine-3, serine-5, and the previously assigned hydroxylation and arabinosylation residue proline-7 were trivial for the endogenous CLV3 function in SAM maintenance. In contrast, substitutions of flanking residues did not impose much damage on CLV3. Complementation of different alanine-substituted constructs was confirmed by measurements of the sizes of SAMs and the WUS expression levels in transgenic plants. These studies established a complete contribution map of individual residues in the peptide-coding region of CLV3 for its function in SAM, which may help to understand peptide hormones in general.

Rhizosphere microbial communities are of great importance to mediate global biogeochemical cycles, plant growth, and fitness. Yet, the processes that drive their assembly remain unclear. The perennial shrubs Caragana spp., which is well known for their role in soil and water conservation, provides an ideal system to study the biogeography of rhizosphere microorganism communities within natural ecosystems. In order to detect how bacterial rhizosphere communities vary in terms of community diversity and composition, the rhizosphere bacterial community of three Caragana species, Caragana microphylla Lam., C. liouana Zhao, and C. korshinskii Kom., which distributed in arid and semi-arid region of northern China were investigated. Across species, Proteobacteria (61.1%), Actinobacteria (16.0%), Firmicutes (8.6%), Bacteroidetes (3.0%), Acidobacteria (3.5%), Gemmatimonadetes (1.4%), and Cyanobacteria (1.0%) were the most dominant phyla in the rhizosphere of the three Caragana species. The relative abundance of Cyanobacteria was significantly higher in rhizosphere of C. korshinskii Kom. compared with C. microphylla Lam. and C. liouana Zhao, while the opposite was found for Gemmatimonadetes in rhizosphere of C. microphylla Lam. relative to C. liouana Zhao. Stepwise multiple linear regression analysis showed that both diversity and richness of the bacterial rhizosphere communities significantly and positively correlated with soil pH (p < 0.01). Distance-based redundancy analysis indicated that soil properties and non-soil parameters detected there accounted for 47.5% of bacterial phylogenetic structure variation (p < 0.01) all together. Meanwhile, soil total phosphorus accounted for the greatest proportion of community structure variance (9.7%, p < 0.01), followed by electrical conduction (6.5%), altitude (5.8%), soil pH (5.4%), mean annual precipitation (3.6%) and total nitrogen (3.6%, p < 0.05 in all cases). Furthermore, partial Mantel test suggested that bacterial rhizosphere community structure significantly correlated with geographical distance, indicating that the less geographical distant sample sites tend to harbor more similar bacterial rhizosphere community. Our results shed new light on the mechanisms of coevolution and interaction between long-lived plants and their rhizosphere bacterial communities across environmental gradients.

Highlight CLE19, as an embryo-expressed CLV3/ESR gene, regulates cotyledon establishment in embryos and nuclear proliferation and cellularization in the endosperm.

Drought is a major environmental constraint affecting growth and distribution of plants in the desert region of the Inner Mongolia plateau. Caragana microphylla, C. liouana, and C. korshinskii are phylogenetically close but distribute vicariously in Mongolia plateau. To gain a better understanding of the ecological differentiation between these three species, we examined the leaf gas exchange, growth, water use efficiency, biomass accumulation and allocation by subjecting their seedlings to low and high drought treatments in a glasshouse. Increasing drought stress had a significant effect on many aspects of seedling performance in all species, but the physiology and growth varied with species in response to drought. C. korshinskii exhibited lower sensitivity of photosynthetic rate and growth, lower specific leaf area, higher biomass allocation to roots, higher levels of water use efficiency to drought compared with the other two species. Only minor interspecific differences in growth performances were observed between C. liouana and C. microphylla. These results indicated that faster seedling growth rate and more efficient water use of C. korshinskii should confer increased drought tolerance and facilitate its establishment in more severe drought regions relative to C. liouana and C. microphylla.

No abstract

BackgroundAlthough it is known that CLAVATA3 (CLV3) acts as 12- and/or 13-amino acid (AA) secreted peptides to regulate the number of stem cells in shoot apical meristems (SAMs), how functional CLV3 peptides are generated and if any particular sequences are required for the processing remain largely unknown.ResultsWe developed a mass spectrometry (MS)-based in vitro assay to monitor the cleavage of heterologously produced CLV3 fusion protein. Through co-cultivation of the fusion protein with Arabidopsis seedlings, we identified two cleavage sites: the previously reported one before Arg70 and a new one before Met39. Using synthetic peptides together with MALDI-Tof-MS analyses, we demonstrated that the non-conserved 5-AA motifs flanking N-termini of the CLV3 and its orthologous CLE1 peptides were critical for their cleavages and optimal activities in vitro. We also found that substitutions of Leu69 by Ala in fusion protein and in synthetic peptide of CLV3 compromised their cleavages, leading to significantly reduced activities in regulating the sizes of shoot and root meristems.ConclusionsThese results suggest that 5-AA residues flanking the N-terminus of CLV3 peptide are required for proper cleavages and optimal function in stem cell regulation.

Leaf stable carbon isotope (δ 13 C) composition provides comprehensive insight into plant carbon cycles and water use efficiency and has also been widely used to evaluate the response of plants to environmental change. In the present study, leaf δ 13 C was analyzed in samples of Caragana microphylla Lam., C. liouana Zhao, and C. korshinskii Kom. from 38 populations. These species provide great environmental benefits and economic value and are distributed east to west continuously across northern China. We studied the relationship of δ 13 C to altitude, mean annual precipitation (MAP), mean annual temperature (MAT), mean annual relative humidity (RH), leaf nitrogen (N), and phosphorus (P) concentrations to examine the patterns and controls of leaf δ 13 C variation in each species. Results indicated that, across the three species, leaf δ 13 C significantly decreased with MAP, RH, and leaf N and P concentrations, while it increased with altitude and MAT. However, patterns and environmental controls of leaf δ 13 C varied proportionally with species. C. korshinskii was mainly controlled by MAP and leaf N concentration, C. liouana was controlled by both MAT and MAP, and C. microphylla was mainly controlled by MAT. Further analysis indicated significant differences in leaf δ 13 C between species, which tended to increase from C. microphylla to C. korshinskii. Overall, these results suggest that the three Caragana species may respond differently to future climate change due to different controlling factors on leaf δ 13 C variation, as well as differentiation in water use efficiency, which likely contributes to the geographical distribution of these species.