POM-POM2/cellulose synthase interacting1 is essential for the functional association of cellulose synthase and microtubules in Arabidopsis.

In plants, regulation of cellulose synthesis is fundamental for morphogenesis and plant growth. Cellulose is synthesized at the plasma membrane, and the orientation of synthesis is guided by cortical microtubules; however, the guiding mechanism is currently unknown. We show that the conditional root elongation pom2 mutants are impaired in cell elongation, fertility, and microtubule-related functions. Map-based cloning of the POM-POM2 locus revealed that it is allelic to CELLULOSE SYNTHASE INTERACTING1 (CSI1). Fluorescently tagged POM2/CSI1s associated with both plasma membrane-located cellulose synthases (CESAs) and post-Golgi CESA-containing compartments. Interestingly, while CESA insertions coincided with cortical microtubules in the pom2/csi1 mutants, the microtubule-defined movement of the CESAs was significantly reduced in the mutant. We propose that POM2/CSI1 provides a scaffold between the CESAs and cortical microtubules that guide cellulose synthesis.

Salinity affects microbial function genes related to nutrient cycling in arid regions.

Introduction: Salinization damages soil system health and influences microbial communities structure and function. The response of microbial functions involved in the nutrient cycle to soil salinization is a valuable scientific question. However, our knowledge of the microbial metabolism functions in salinized soil and their response to salinity in arid desert environments is inadequate. Methods: Here, we applied metagenomics technology to investigate the response of microbial carbon (C), nitrogen (N), phosphorus (P), and sulfur (S) cycling and the key genes to salinity, and discuss the effects of edaphic variables on microbial functions. Results: We found that carbon fixation dominated the carbon cycle. Nitrogen fixation, denitrification, assimilatory nitrate reduction (ANRA), and nitrogen degradation were commonly identified as the most abundant processes in the nitrogen cycle. Organic phosphorus dissolution and phosphorus absorption/transport were the most enriched P metabolic functions, while sulfur metabolism was dominated by assimilatory sulfate reduction (ASR), organic sulfur transformation, and linkages between inorganic and organic sulfur transformation. Increasing salinity inhibited carbon degradation, nitrogen fixation, nitrogen degradation, anammox, ANRA, phosphorus absorption and transport, and the majority of processes in sulfur metabolism. However, some of the metabolic pathway and key genes showed a positive response to salinization, such as carbon fixation (facA, pccA, korAB), denitrification (narG, nirK, norBC, nosZ), ANRA (nasA, nirA), and organic phosphorus dissolution processes (pstABCS, phnCD, ugpAB). High salinity reduced the network complexity in the soil communities. Even so, the saline microbial community presented highly cooperative interactions. The soil water content had significantly correlations with C metabolic genes. The SOC, N, and P contents were significantly correlated with C, N, P, and S network complexity and functional genes. AP, NH4+, and NO3- directly promote carbon fixation, denitrification, nitrogen degradation, organic P solubilization and mineralization, P uptake and transport, ASR, and organic sulfur transformation processes. Conclusion: Soil salinity in arid region inhibited multiple metabolic functions, but prompted the function of carbon fixation, denitrification, ANRA, and organic phosphorus dissolution. Soil salinity was the most important factor driving microbial functions, and nutrient availability also played important roles in regulating nutrient cycling.

The developing xylem transcriptome and genome-wide analysis of alternative splicing in Populus trichocarpa (black cottonwood) populations.

BACKGROUND: Alternative splicing (AS) of genes is an efficient means of generating variation in protein structure and function. AS variation has been observed between tissues, cell types, and different treatments in non-woody plants such as Arabidopsis thaliana (Arabidopsis) and rice. However, little is known about AS patterns in wood-forming tissues and how much AS variation exists within plant populations. RESULTS: Here we used high-throughput RNA sequencing to analyze the Populus trichocarpa (P. trichocarpa) xylem transcriptome in 20 individuals from different populations across much of its range in western North America. Deep transcriptome sequencing and mapping of reads to the P. trichocarpa reference genome identified a suite of xylem-expressed genes common to all accessions. Our analysis suggests that at least 36% of the xylem-expressed genes in P. trichocarpa are alternatively spliced. Extensive AS was observed in cell-wall biosynthesis related genes such as glycosyl transferases and C2H2 transcription factors. 27902 AS events were documented and most of these events were not conserved across individuals. Differences in isoform-specific read densities indicated that 7% and 13% of AS events showed significant differences between individuals within geographically separated southern and northern populations, a level that is in general agreement with AS variation in human populations. CONCLUSIONS: This genome-wide analysis of alternative splicing reveals high levels of AS in P. trichocarpa and extensive inter-individual AS variation. We provide the most comprehensive analysis of AS in P. trichocarpa to date, which will serve as a valuable resource for the plant community to study transcriptome complexity and AS regulation during wood formation.

Gene expression patterns underlying changes in xylem structure and function in response to increased nitrogen availability in hybrid poplar.

Nitrogen availability has a strong influence on plant growth and development. In this study, we examined the effect of nitrogen availability on xylogenesis in hybrid poplar (Populus trichocarpa x deltoides H11-11). Saplings of hybrid poplar were fertilized for 33 d with either high or adequate levels of ammonium nitrate. We observed enhanced radial growth, wider vessels and fibres and thinner fibre walls in the secondary xylem of high N relative to adequate N plants. These anatomical differences translated into altered hydraulic properties with xylem being more transport efficient but also more vulnerable to drought-induced cavitation in high N plants. The changes in xylem structure and function were associated with differences in gene expression as revealed by the transcriptome analysis of the developing xylem region. We found 388 genes differentially expressed (fold change ±1.5, P-value ≤ 0.05), including a number of genes putatively involved in nitrogen and carbohydrate metabolism and various aspects of xylem cell differentiation. Several genes encoding known transcriptional regulators of secondary cell wall deposition were down-regulated in high N plants, corresponding with thinner secondary cell walls in these plants. The results of this study provide us with gene candidates potentially affecting xylem hydraulic and structural traits.

Structural Characteristics and Assembly Mechanisms of Soil Microbial Communities under Water-Salt Gradients in Arid Regions.

Exploring the structural characteristics of arid soil microbial communities and their assembly mechanisms is important for understanding the ecological characteristics of arid zone soils and promoting ecological restoration. In this study, we used Illumina high-throughput sequencing technology to study soils in the arid zone of the Lake Ebinur basin, determined the differences among soil microbial community structures in the study area under different water-salt gradients, and investigated the effects of environmental factors on microbial community structure and assembly mechanisms. The results show the following: the microbial community alpha diversity exhibited a significantly higher low water-salt gradient (L) than high water-salt gradient (H) and medium water-salt gradient (M). The pH was most strongly correlated with soil microbial community structure, where the alpha diversity indices of the bacterial community and fungal community were significantly negatively correlated with pH, and the Bray-Curtis distance of bacterial community was significantly positively correlated with pH (p < 0.05). The complexity of bacterial community co-occurrence networks showed a significantly higher L than H and M, and the complexity of fungal community co-occurrence network showed a significantly lower L than H and M. The cooperative relationship of H and M in the co-occurrence networks was stronger than that of the L, and the key species of the microbial co-occurrence network were different under different water-salt gradients. Stochastic processes dominated the assembly mechanism of the microbial community structure of soil, and the explanation rates of deterministic and stochastic processes were different under different water-salt gradients, with the highest explanation rate of stochastic processes on the L accounting for more than 90%. In summary, the soil microbial community structure and assembly mechanisms significantly differed across water-salt gradients, and these findings can help provide a reference for further research on soil microbiology in arid zones.

Regulation of secondary growth by poplar BLADE-ON-PETIOLE genes in Arabidopsis.

BLADE-ON-PETIOLE (BOP) genes are essential regulators of vegetative and reproductive development in land plants. First characterized in Arabidopsis thaliana (Arabidopsis), members of this clade function as transcriptional co-activators by recruiting TGACG-motif binding (TGA) basic leucine zipper (bZIP) transcription factors. Highly expressed at organ boundaries, these genes are also expressed in vascular tissue and contribute to lignin biosynthesis during secondary growth. How these genes function in trees, which undergo extensive secondary growth to produce wood, remains unclear. Here, we investigate the functional conservation of BOP orthologs in Populus trichocarpa (poplar), a widely-used model for tree development. Within the poplar genome, we identified two BOP-like genes, PtrBPL1 and PtrBPL2, with abundant transcripts in stems. To assess their functions, we used heterologous assays in Arabidopsis plants. The promoters of PtrBPL1 and PtrBPL2, fused with a ß-glucuronidase (GUS) reporter gene showed activity at organ boundaries and in secondary xylem and phloem. When introduced into Arabidopsis plants, PtrBPL1 and PtrBPL2 complemented leaf and flower patterning defects in bop1 bop2 mutants. Notably, Arabidopsis plants overexpressing PtrBPL1 and PtrBPL2 showed defects in stem elongation and the lignification of secondary tissues in the hypocotyl and stem. Finally, PtrBPL1 and PtrBPL2 formed complexes with TGA bZIP proteins in yeast. Collectively, our findings suggest that PtrBPL1 and PtrBPL2 are orthologs of Arabidopsis BOP1 and BOP2, potentially contributing to secondary growth regulation in poplar trees. This work provides a foundation for functional studies in trees.

OVATE FAMILY PROTEIN4 (OFP4) interaction with KNAT7 regulates secondary cell wall formation in Arabidopsis thaliana.

The homeodomain transcription factor KNAT7 has been reported to be involved in the regulation of secondary cell wall biosynthesis. Previous work suggested that KNAT7 can interact with members of the Ovate Family Protein (OFP) transcription co-regulators. However, it remains unknown whether such an OFP-KNAT7 complex could be involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. We re-tested OFP1 and OFP4 for their abilities to intact with KNAT7 using yeast two-hybrid assays, and verified KNAT7-OFP4 interaction but found only weak interaction between KNAT7 and OFP1. Further, the interaction of KNAT7 with OFP4 appears to be mediated by the KNAT7 homeodomain. We used bimolecular fluorescence complementation to confirm interactions and found that OFP1 and OFP4 both interact with KNAT7 in planta. Using a protoplast transient expression system we showed that KNAT7 as well as OFP1 and OFP4 act as transcriptional repressors. Furthermore, in planta interactions between KNAT7 and both OFP1 and OFP4 enhance KNAT7's transcriptional repression activity. An ofp4 mutant exhibited similar irx and fiber cell wall phenotypes as knat7, and the phenotype of a double ofp4 knat7mutant was similar to those of the single mutants, consistent with the view that KNAT7 and OFP function in a common pathway or complex. Furthermore, the pleiotropic OFP1 and OFP4 overexpression phenotype was suppressed in a knat7 mutant background, suggesting that OFP1 and OFP4 functions depend at least partially on KNAT7 function. We propose that KNAT7 forms a functional complex with OFP proteins to regulate aspects of secondary cell wall formation.

The Class II KNOX gene KNAT7 negatively regulates secondary wall formation in Arabidopsis and is functionally conserved in Populus.

• The formation of secondary cell walls in cell types such as tracheary elements and fibers is a defining characteristic of vascular plants. The Arabidopsis transcription factor KNAT7 is a component of a transcription network that regulates secondary cell wall biosynthesis, but its function has remained unclear. • We conducted anatomical, biochemical and molecular phenotypic analyses of Arabidopsis knat7 loss-of-function alleles, KNAT7 over-expression lines and knat7 lines expressing poplar KNAT7. • KNAT7 was strongly expressed in concert with secondary wall formation in Arabidopsis and poplar. Arabidopsis knat7 loss-of-function alleles exhibited irregular xylem phenotypes, but also showed increased secondary cell wall thickness in fibers. Increased commitment to secondary cell wall biosynthesis was accompanied by increased lignin content and elevated expression of secondary cell wall biosynthetic genes. KNAT7 over-expression resulted in thinner interfascicular fiber cell walls. • Taken together with data demonstrating that KNAT7 is a transcriptional repressor, we hypothesize that KNAT7 is a negative regulator of secondary wall biosynthesis, and functions in a negative feedback loop that represses metabolically inappropriate commitment to secondary wall formation, thereby maintaining metabolic homeostasis. The conservation of the KNAT7 regulatory module in poplar suggests new ways to manipulate secondary cell wall deposition for improvement of bioenergy traits in this tree.

AtMYB61, an R2R3-MYB transcription factor, functions as a pleiotropic regulator via a small gene network.

Throughout their lifetimes, plants must coordinate the regulation of various facets of growth and development. Previous evidence has suggested that the Arabidopsis thaliana R2R3-MYB, AtMYB61, might function as a coordinate regulator of multiple aspects of plant resource allocation. Using a combination of cell biology, transcriptome analysis and biochemistry, in conjunction with gain-of-function and loss-of-function genetics, the role of AtMYB61 in conditioning resource allocation throughout the plant life cycle was explored. In keeping with its role as a regulator of resource allocation, AtMYB61 is expressed in sink tissues, notably xylem, roots and developing seeds. Loss of AtMYB61 function decreases xylem formation, induces qualitative changes in xylem cell structure and decreases lateral root formation; in contrast, gain of AtMYB61 function has the opposite effect on these traits. AtMYB61 coordinates a small network of downstream target genes, which contain a motif in their upstream regulatory regions that is bound by AtMYB61, and AtMYB61 activates transcription from this same motif. Loss-of-function analysis supports the hypothesis that AtMYB61 targets play roles in shaping subsets of AtMYB61-related phenotypes. Taken together, these findings suggest that AtMYB61 links the transcriptional control of multiple aspects of plant resource allocation.

Regulation of secondary cell wall biosynthesis by poplar R2R3 MYB transcription factor PtrMYB152 in Arabidopsis.

Poplar has 192 annotated R2R3 MYB genes, of which only three have been shown to play a role in the regulation of secondary cell wall formation. Here we report the characterization of PtrMYB152, a poplar homolog of the Arabidopsis R2R3 MYB transcription factor AtMYB43, in the regulation of secondary cell wall biosynthesis. The expression of PtrMYB152 in secondary xylem is about 18 times of that in phloem. When expressed in Arabidopsis under the control of either 35S or PtrCesA8 promoters, PtrMYB152 increased secondary cell wall thickness, which is likely caused by increased lignification. Accordingly, elevated expression of genes encoding sets of enzymes in secondary wall biosynthesis were observed in transgenic plants expressing PtrMYB152. Arabidopsis protoplast transfection assays suggested that PtrMYB152 functions as a transcriptional activator. Taken together, our results suggest that PtrMYB152 may be part of a regulatory network activating expression of discrete sets of secondary cell wall biosynthesis genes.

Global transcript profiling of primary stems from Arabidopsis thaliana identifies candidate genes for missing links in lignin biosynthesis and transcriptional regulators of fiber differentiation.

Different stages of vascular and interfascicular fiber differentiation can be identified along the axis of bolting stems in Arabidopsis. To gain insights into the metabolic, developmental, and regulatory events that control this pattern, we applied global transcript profiling employing an Arabidopsis full-genome longmer microarray. More than 5000 genes were differentially expressed, among which more than 3000 changed more than twofold, and were placed into eight expression clusters based on polynomial regression models. Within these, 182 upregulated transcription factors represent candidate regulators of fiber development. A subset of these candidates has been associated with fiber development and/or secondary wall formation and lignification in the literature, making them targets for functional studies and comparative genomic analyses with woody plants. Analysis of differentially expressed phenylpropanoid genes identified a set known to be involved in lignin biosynthesis. These were used to anchor co-expression analyses that allowed us to identify candidate genes encoding proteins involved in monolignol transport and monolignol dehydrogenation and polymerization. Similar analyses revealed candidate genes encoding enzymes that catalyze missing links in the shikimate pathway, namely arogenate dehydrogenase and prephenate aminotransferase.