Transcription factor NTL9 negatively regulates Arabidopsis vascular cambium development during stem secondary growth

Abstract In plant stems, secondary vascular development is established through the differentiation of cylindrical vascular cambium, producing secondary xylem (wood) and phloem (bast), which have economic importance. However, there is a dearth of knowledge on the genetic mechanism underlying this process. NAC with Transmembrane Motif 1-like transcription factor 9 (NTL9) plays a central role in abiotic and immune signaling responses. Here, we investigated the role of NTL9 in vascular cambium development in Arabidopsis (Arabidopsis thaliana) inflorescence stems by identifying and characterizing an Arabidopsis phloem circular-timing (pct) mutant. The pct mutant exhibited enhanced vascular cambium formation following secondary phloem production. In the pct mutant, although normal organization in vascular bundles was maintained, vascular cambium differentiation occurred at an early stage of stem development, which was associated with increased expression of cambium-/phloem-related genes and enhanced cambium activity. The pct mutant stem phenotype was caused by a recessive frameshift mutation that disrupts the transmembrane (TM) domain of NTL9. Our results indicate that NTL9 functions as a negative regulator of cambial activity and has a suppressive role in developmental transition to the secondary growth phase in stem vasculature, which is necessary for its precise TM domain-mediated regulation.

without primers. The first PCR product was used as a template for the second PCR using primers P_115 and P_1528. The resulting DNA fragment was inserted into the BamHI/SacI sites of the pBI121 vector (Clontech, Mountain View, CA) using the In-Fusion Cloning System to produce p35Spro:GFP-RCI2a. The modified DNA fragment containing the GFP-RCI2a translational fusion region was obtained using PCR with p35Spro:GFP-RCI2a as a template and primers P_1531 and P_1528. This PCR fragment was inserted into the SalI/SacI sites of pSUC2pro:GUS using the In-Fusion Cloning System.
To construct a plasmid expressing the GUS reporter gene under the control of the NTL9 promoter (pNTL9pro:GUS), a DNA fragment containing the NTL9 promoter was obtained using PCR with primers P_1393 and P_1394. This PCR fragment was inserted into the SalI/BamHI sites of the pAtPP2CF1: GUS plasmid (Sugimoto et al., 2014) using the In-Fusion Cloning System.
To construct a plasmid possessing the NTL9 genomic region containing both the NTL9 promoter and the entire NTL9 gene including introns (pNTL9pro:NTL9g), a DNA fragment containing the NTL9 genomic region was obtained using PCR with primers P_1393 and P_1395. This PCR fragment was inserted into the SalI/EcoRI sites of the pAtPP2CF1:GUS plasmid (Sugimoto et al., 2014) using the In-Fusion Cloning System.
To construct a plasmid expressing the entire NTL9 gene, including introns, under the control of the 35S promoter (p35Spro:NTL9g), two sequential PCRs were performed to produce the modified DNA fragment containing the NTL9 genome. The first PCR was performed using primers P_1407 and P_1408. The first PCR product was used as a template for the second PCR using primers P_115 and P_1408. The resulting DNA fragment containing the NTL9 genome was inserted into the BamHI/SacI sites of the pBI121 vector using the In-Fusion Cloning System.
To construct a plasmid expressing NTL9.1 cDNA under the control of the 35S promoter (p35Spro:NTL9.1c), a DNA fragment containing the NTL9.1 cDNA was obtained using PCR with primers P_1400 and P_1401. This PCR fragment was inserted into the pGEM-T Easy vector to produce pNTL9.1c. Two sequential PCRs were performed to produce the modified DNA fragment containing the NTL9.1 cDNA. The first PCR was performed using pNTL9.1c as a template with primers P_1407 and P_1408. The first PCR product was used as a template for the second PCR using primers P_115 and P_1408. The resulting DNA fragment containing the NTL9.1 cDNA was inserted into the BamHI/SacI sites of the pBI121 vector using the In-Fusion Cloning System.

Supplemental Method S2.
Map-based cloning of the PCT gene.
F2 populations derived from crosses between the pct mutant and wild-type Landsberg erecta (Ler)-0 were used for genetic linkage mapping of the PCT locus. The PCT gene was mapped using sets of cleaved amplified polymorphic sequence (CAPS) and simple sequence length polymorphic (SSLP) markers listed in Supplemental Table S4. To identify the pct mutation, the genomic region around the PCT locus was compared between wild-type and pct mutants using whole-genome sequencing with next-generation sequencing, and one base insertion (a nucleotide A) in the coding region of NTL9 (At4G35580) was identified. Cross-sections were 100-μm-thick and stained with toluidine blue. Ca, cambium; Ph, phloem; Xy, xylem. Scale bars, 100 μm.    Table S3). The nucleotide position on chromosome 4 is indicated in parenthesis. To identify the PCT gene, we mapped PCT to chromosome 4, delimited to a locus within a 500-kb region between the markers Chr.4_16.7 Mb and Chr.4_17.2 Mb using 72 F2 chromosomes. The values on the right are the numbers of recombinants in the intervals between the PCT locus and each marker. Supplemental a +/-indicate plants with or without the transgene, respectively. b χ 2 value for wild-type : mutant stem phenotype segregation of 3 : 1 was calculated using two phenotypic classes (one degree of freedom). The 95% confidence limit for rejecting the expected 3 : 1 segregation is ≥3.84 and the 99% limit is ≥6.63.