SHORT-ROOT paralogs mediate feedforward regulation of D-type cyclin to promote nodule formation in soybean.

Nitrogen fixation in soybean takes place in root nodules that arise from de novo cell divisions in the root cortex. Although several early nodulin genes have been identified, the mechanism behind the stimulation of cortical cell division during nodulation has not been fully resolved. Here we provide evidence that two paralogs of soybean SHORT-ROOT (GmSHR) play vital roles in soybean nodulation. Expression of GmSHR4 and GmSHR5 (GmSHR4/5) is induced in cortical cells at the beginning of nodulation, when the first cell divisions occur. The expression level of GmSHR4/5 is positively associated with cortical cell division and nodulation. Knockdown of GmSHR5 inhibits cell division in outer cortical layers during nodulation. Knockdown of both paralogs disrupts the cell division throughout the cortex, resulting in poorly organized nodule primordia with delayed vascular tissue formation. GmSHR4/5 function by enhancing cytokinin signaling and activating early nodulin genes. Interestingly, D-type cyclins act downstream of GmSHR4/5, and GmSHR4/5 form a feedforward loop regulating D-type cyclins. Overexpression of D-type cyclins in soybean roots also enhanced nodulation. Collectively, we conclude that the GmSHR4/5-mediated pathway represents a vital module that triggers cytokinin signaling and activates D-type cyclins during nodulation in soybean.

Intersected functional zone of transcriptional regulators patterns stemness within stem cell niche of root apical meristem.

Root stem cell niche (SCN) consists of a quiescent center (QC) and surrounding stem cells. Disrupted symplastic communication leads to loss of stemness in the whole SCN. Several SCN regulators were reported to move between cells for SCN maintenance. However, single mutant of these regulators is insufficient to abolish QC stemness despite the high differentiation rate in surrounding stem cells. To dissect the mechanism behind such distinct stemness in SCN, we combined the mis-expression strategy with pWOX5:icals3m system in which QC is symplastically isolated. We found the starch accumulation in QC could be synergistically repressed by WUSCHEL-RELATED HOMEOBOX 5 (WOX5), SHORT-ROOT (SHR), SCARCROW (SCR), and PLETHORA (PLT). Like PLTs, other core regulators also exhibited dimorphic functions by inhibiting differentiation at a higher dose while promoting cell division at a low protein level. Being located in the center of the intersected expression zones, QC cells receive the highest level of core regulators, forming the most robust stemness within SCN. WUSCHEL-RELATED HOMEOBOX 5 was sufficient to activate PLT1/2 expression, contributing to the QC-enriched PLTs. Our results provide experimental evidence supporting the long-standing hypothesis that the combination of spatial expression, synergistic function and dosage effect of core regulators result in spatially distinct stemness in SCN.

Cell-Fate Specification in Arabidopsis Roots Requires Coordinative Action of Lineage Instruction and Positional Reprogramming.

Tissue organization and pattern formation within a multicellular organism rely on coordinated cell division and cell-fate determination. In animals, cell fates are mainly determined by a cell lineage-dependent mechanism, whereas in plants, positional information is thought to be the primary determinant of cell fates. However, our understanding of cell-fate regulation in plants mostly relies on the histological and anatomical studies on Arabidopsis (Arabidopsis thaliana) roots, which contain a single layer of each cell type in nonvascular tissues. Here, we investigate the dynamic cell-fate acquisition in modified Arabidopsis roots with additional cell layers that are artificially generated by the misexpression of SHORT-ROOT (SHR). We found that cell-fate determination in Arabidopsis roots is a dimorphic cascade with lineage inheritance dominant in the early stage of pattern formation. The inherited cell identity can subsequently be removed or modified by positional information. The instruction of cell-fate conversion is not a fast readout during root development. The final identity of a cell type is determined by the synergistic contribution from multiple layers of regulation, including symplastic communication across tissues. Our findings underline the collaborative inputs during cell-fate instruction.

Symplastic communication spatially directs local auxin biosynthesis to maintain root stem cell niche in Arabidopsis.

Stem cells serve as the source of new cells for plant development. A group of stem cells form a stem cell niche (SCN) at the root tip and in the center of the SCN are slowly dividing cells called the quiescent center (QC). QC is thought to function as a signaling hub that inhibits differentiation of surrounding stem cells. Although it has been generally assumed that cell-to-cell communication provides positional information for QC and SCN maintenance, the tools for testing this hypothesis have long been lacking. Here we exploit a system that effectively blocks plasmodesmata (PD)-mediated signaling to explore how cell-to-cell communication functions in the SCN. We showed that the symplastic signaling between the QC and adjacent cells directs the formation of local auxin maxima and establishment of AP2-domain transcription factors, PLETHORA gradients. Interestingly we found symplastic signaling is essential for local auxin biosynthesis, which acts together with auxin polar transport to provide the guidance for local auxin enrichment. Therefore, we demonstrate the crucial role of cell-to-cell communication in the SCN maintenance and further uncover a mechanism by which symplastic signaling initiates and reinforces the positional information during stem cell maintenance via auxin regulation.

Pseudomonas aeruginosa phage PaP1 DNA polymerase is an A-family DNA polymerase demonstrating ssDNA and dsDNA 3'-5' exonuclease activity.

Most phages contain DNA polymerases, which are essential for DNA replication and propagation in infected host bacteria. However, our knowledge on phage-encoded DNA polymerases remains limited. This study investigated the function of a novel DNA polymerase of PaP1, which is the lytic phage of Pseudomonas aeruginosa. PaP1 encodes its sole DNA polymerase called Gp90 that was predicted as an A-family DNA polymerase with polymerase and 3'-5' exonuclease activities. The sequence of Gp90 is homologous but not identical to that of other A-family DNA polymerases, such as T7 DNA polymerases (Pol) and DNA Pol I. The purified Gp90 demonstrated a polymerase activity. The processivity of Gp90 in DNA replication and its efficiency in single-dNTP incorporation are similar to those of T7 Pol with processive thioredoxin (T7 Pol/trx). Gp90 can degrade ssDNA and dsDNA in 3'-5' direction at a similar rate, which is considerably lower than that of T7 Pol/trx. The optimized conditions for polymerization were a temperature of 37 °C and a buffer consisting of 40 mM Tris-HCl (pH 8.0), 30 mM MgCl2, and 200 mM NaCl. These studies on DNA polymerase encoded by PaP1 help advance our knowledge on phage-encoded DNA polymerases and elucidate PaP1 propagation in infected P. aeruginosa.

[Effect of endophytic fungi on expression amount of key enzyme genes in saponins biosynthesis and Eleutherococcus senticosus saponins content].

OBJECTIVE: To analyze the effect of endophytic fungi on expression amount of key enzyme genes SS (squalene synthase gene), SE (squalene epoxidase gene) and bAS (beta-amyrin synthase gene) in saponin biosynthesis and saponins content in Eleutherococcus senticosus. METHOD: Wound method was used for back meeting the endophytic fungi to E. senticosus. With GAPDH as internal control gene, the expression of key enzyme genes was detected by real time PCR method. E. senticosus saponins content was measured by spectrophotometry method. RESULT: When wound method back meeting P116-1a and P116-1b after 30 d, the expression content of SS improved significantly (P < 0.05), however the back meeting of P109-4 and P312-1 didnt change the expression of SS. After that SS expression showed reduction-equality-reduction varying trend. Thirty days after back meeting P312-1, the expression content of SE improved significantly (P < 0.05). Ninty days after back meeting P116-1b and P312-1, the expression content of SE improved significantly to 130%,161%, respectively (P < 0.05). After 120 d, back meeting four endophytic fungi, the expression of SE were significantly higher than the control (P < 0.05). Back meeting four endophytic fungi form 60 d to 120 d, the expression of bAS was significantly higher than the control (P < 0.05). The back meeting four endophytic fungi improved E. senticosus saponins content significantly (P < 0.05). CONCLUSION: Endophytic fungi P116-1a, P116-1b, P1094 and P312-1 significantly effected the expression of key enzyme genes SS, SE and bAS and then affected E. senticosus saponins content. Among the genes, bAS was key target gene.

[Molecular cloning of farnesyl diphosphate synthase from Eleutherococcus senticosus and its bioinformatics and expression analysis].

OBJECTIVE: To clone farnesyl diphosphate synthase (FPS) gene from Eleutherococcus senticosus and analyze the bioinformatics and expression of the gene. METHOD: The FPS full length cDNA was cloned by rapid amplification of cDNA ends (RACE). The data was analyzed by bioinformatics method, the structure and function of FPS was deduced. The expression of FPS in different organ of E. senticosus was detected by RT-PCR. RESULT: The full length of FPS cDNA was 1 499 bp containing a 1 029 bp ORF that encoded 342 amino acids. The deduced protein sequence exhibited two Asp riches conserved motifs (DDXXD). Without transmembrane domain, FPS was located in cytoplasm. RT-PCR result showed that FPS gene expressed in different organs of E. senticosus. The expression amounts of FPS in different organs were different significantly (P < 0.05). CONCLUSION: The FPS gene of E. senticosus was successfully cloned for the first time, and provided a stable foundation for studying on its effect and expression control on E. senticosus saponins biosynthesis.

[Analyze on the expression differences of key genes in saponins biosynthesize in Eleutherococcus senticosus and its callus].

OBJECTIVE: To analyze the expression differences of SS, SE and bAS in leaf, petiole and callus from leaf and petiole of Eleutherococcus senticosus. METHODS: Calluses were induced from explants of E. senticosus leaf and petiole. The expression amount of SS, SE and bAS gene was detected by real time PCR. RESULTS: The expression amount of SS, SE and bAS genes in callus from leaf were 89.3%, 73.8% and 83.4% of that in leaf, respectively. The expression amount of SS, SE and bAS genes in callus from petiole were 80.2%, 87.7% and 70.9% of that in petiole, respectively. CONCLUSION: The expression amount of SS, SE and bAS gene in callus from E. senticosus are significantly lower than that in plant.