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A Phytochrome B-PIF4-MYC2 Module Tunes Secondary Cell Wall Thickening in Response to Shaded Lighting

Secondary cell walls (SCW) in stem xylem cells provide mechanical strength and structural support for growth. SCW thickening is light- regulated and varies under different light growth conditions. Our previous study revealed that blue light enhances SCW thickening through the activity of MYC2 directed by CRYPTOCHROME1 (CRY1) signaling in stem xylary fiber cells. In this study, we demonstrate that the low ratio of red: far-red light (R:FR) of the shaded light condition inhibits SCW thickening in the inflorescence stem of Arabidopsis. Phytochrome B (PHYB) plays a dominant role in perceiving the R:FR balance. Under white and red-light conditions, phyB mutants display thinner SCWs in xylary fibers, but thicker SCWs are deposited in the PHYTOCHROME INTERACTING FACTORS (PIFs) quadruple mutant pif1pif3pif4pif5 (pifq), suggesting involvement of the PHYB-PIFs signaling module in regulating SCW thickening. Interaction of PIF4 with MYC2 affects MYC2 localization in nuclei and inhibits its transactivation activity on the NST1 promoter. Shade conditions mediate the PIF4 interaction with MYC2 to regulate SCW thickening. Genetic analysis confirms that the regulation of SCW thickening by PIFs is dependent on MYC2 function. Together, these data reveal a molecular mechanism for the effect of shaded light inhibition on SCW thickening in stems of Arabidopsis.

. To dissect the 135 genetic base of the light R:FR ratio effect on SCW thickening, we analyzed the 136 inflorescence stem growth of phyB and quadruple pif1pif3pif4pif5 (pifq) mutants.  Figure S4A). Also, PIF4 promoter-GUS analysis indicated its 192 expression in interfascicular fiber cells (Supplemental Figure S4B). 193 Next, we dissected the molecular pathway that mediates the PHYB-PIFs signal to  Table S1). The red-light induction of NST1, MYC2 and other key 211 genes for SCW thickening was further confirmed in RT-qPCR analysis 212 (Supplemental Figure S6). Conversely, when exposed to far-red light conditions,

219
It is known that MYC2 binds to the NST1 promoter to regulate SCW   (Figure 1, D and E). Genetic evidence reveals that the PHYB-PIFs 326 interaction mediates the signaling underlying the low R:FR ratio inhibition of SCW 327 thickening in fiber cells but not in vessel cells (Figure 2). Interestingly, the blue light  Table S1). Therefore, light-induced SCW thickening appears to be through the 332 transcriptional network directed by NST1 in fiber rather than xylem cells.  (Figure 6, A and B). 350 Therefore, we conclude that the PHYB-PIFs signaling module affects MYC2 protein 351 stability.

352
PIFs interact with MYC2 and such interaction plays a role in regulating the NST1 353 promoter transcriptional activity (Figure 5, A and B). MYC2/MYC4 activates NST1 354 transcription by binding its promoter, but PIF4 is inactive to NST1 transcription.         A, Growth of Arabidopsis inflorescence stems in WL and WL+FR conditions. WL: white light. FR: far red. Scale bar = 5 cm. B, Elongation of inflorescence stems in WL and WL+FR conditions during growth. n = 9, mean ± SD. C, Tensile strength of the inflorescence stems. Student's t test (**P < 0.01) was used for statistical analysis, n = 18, mean ± SD. D, Cross sections of the inflorescence stem grown under different light conditions (WL and WL+FR) visualized under the light microscope (after Toluidine blue staining; LHS panels) and the transmission electron microscope (RHS panels). If: Interfascicular fiber cell. V: Vessel cell. LHS panels: scale bar = 20 μm, RHS panels: scale bar = 5 μm. E, Measurements of SCW thickness in the interfascicular fiber cells in (D). From three biological replicates, more than 10 cells were measured per biological replicate. Student's t test (**P < 0.01) was used for statistical analysis, mean ± SD. F, Lignin content in inflorescence stems of the plants grown in different R:FR conditions. Student's t test (*P < 0.05) was used for statistical analyses, n = 3, mean ± SD. G, Crystalline cellulose content in inflorescence stems of the plants grown in different R:FR conditions. Student's t test (*P < 0.05) was used for statistical analyses, n = 3, mean ± SD.

Figure 2 PHYB and PIFs regulate SCW thickening in fiber cells.
A, Plants were grown in white light (WL) at 8-weeks old. Scale bar = 5 cm. B, Measurements of plant height in (A). Student's t test (**P < 0.01) was used for statistical analyses, n = 20, mean ± SD. C,Inflorescence stem elongation. The stem was marked with two points at the basal region and the distance between the two points was measured every day during growth. Student's t test (*P < 0.05) was used for statistical analyses, n = 3, mean ± SD. D, Fiber cell length measured in disaggregated fiber cells. Data were from three biological replicates and more than 200 cells were measured per biological repeats. Student's t test (**P < 0.01) was used for statistical analysis, mean ± SD. E, Transmission electron micrographs of inflorescence stem cross-sections. If: Interfascicular fiber cell. V: Vessel cell. Scale bar = 5 μm. F, Measurements of SCW thickness in the interfascicular fiber cells and vessel cells in (E). Data were from three biological replicates and more than 5 cells were measured per biological replicate. Student's t test (**P < 0.01) was used for statistical analysis, mean ± SD. G, Tensile strength measurements of the inflorescence stem. Student's t test (**P < 0.01) was used for statistical analysis, n = 16, mean ± SD. H, Lignin content in inflorescence stems. Student's t test (**P < 0.01, *P < 0.05) was used for statistical analyses, n = 3, mean ± SD. I, Crystalline cellulose content in inflorescence stems. Student's t test (**P < 0.01, *P < 0.05) was used for statistical analyses, n = 3, mean ± SD.

Figure 3 SCW phenotypes of PHYB-OE and PIF4-OE plants.
A, Transmission electron micrographs of inflorescence stem cross-sections. If: Interfascicular fiber cell. V: Vessel cells. Scale bar = 5 μm. B, Measurements of SCW thickness of cells in (A). Data were from three biological replicates and more than 10 cells were measured per biological replicate. Student's t test (**P < 0.01) was used for statistical analysis, mean ± SD. C, Tensile strength of inflorescence stems. Student's t test (**P < 0.01) was used for statistical analysis, n = 15, mean ± SD. D, Lignin content in inflorescence stems. Student's t test (*P < 0.05) was used for statistical analyses, n = 3, mean ± SD. E, Cellulose content in inflorescence stems. Student's t test (**P < 0.01, *P < 0.05) was used for statistical analyses, n = 3, mean ± SD. F, Expression of SCW regulatory (NST1 & SND1) and biosynthesis-related (4CL1 & IRX8) genes in different genotypes grown under white light. Three biological repeats were performed. Student's t test (**P < 0.01, *P < 0.05) was used for statistical analysis, mean ± SD.

PIFs.
A, The inflorescence stems of phyB and pifq mutants were grown under red light (high R:FR) and anatomically analyzed. Transmission electron micrographs of the inflorescence stem cross-sections. If: Interfascicular fiber cell. V: Vessel. Scale bar = 5 μm. B, Statistics of SCW thickness in (A). Data were three biological replicates and more than 10 cells were measured per biological replicate. Student's t test (**P < 0.01) was used for statistical analysis, mean ± SD. C, Tensile strength of the inflorescence stem. Student's t test (**P < 0.01) was used for statistical analysis, n = 20, mean ± SD. D, Lignin content in the inflorescence stem. Student's t test (**P < 0.01, *P < 0.05) was used for statistical analyses, n = 3, mean ± SD. E, Expression of SCW regulatory (NST1) and biosynthesis-related (CESA4, 4CL1 & IRX8) was measured by qRT-PCR analysis. Three biological replicates were performed. Student's t test (**P < 0.01) was used for statistical analysis, mean ± SD. A, Schematic representation of the NST1 promoter-driven dual-LUC reporter gene and three effector gene constructs. 35S promoter, NST1 promoter (-1 to -3711 bp from ATG), renilla luciferase (REN), firefly luciferase (LUC) are indicated in reporter constructs. In effector constructs, PIF4, PIF5 and MYC2 are driven by the 35S promoter. B, PIF4/PIF5 inhibit MYC2 activation of NST1 promoter. Arabidopsis protoplasts were transfected with the reporter constructs in combination with different effector consrtructs. After transfection, the protoplasts were kept in dark for 16 h. Relative luminescence was normalized to the protoplast transformed with reporter and empty effector (GFP). Student's t test (**P < 0.01) was used for statistical analysis, n = 3, mean ± SD. C, Subcellular localization of PIF4 and MYC2/MYC4. Constructs of PIF4-CFP, MYC2-YFP and MYC4-YFP were transferred to tobacco leaves, separately or together, by Agro-infiltration. Then the tobacco leaves were kept in dark for 12 h before fluorescence observation. Scale bar = 5 μm A, Immunoblot of MYC2-YFP and ACTIN in transgenic plants. Seedlings were grown in WL condition for 10 days and then treated with dark condition. Similar results were observed in three independent experiments. B, Immunoblot of MYC2-YFP and ACTIN in transgenic plants. Seedlings were grown in WL condition for 10 days and then treated with far-red light. Similar results were observed in three independent experiments. C, Fluorescence signals in seedling roots. Seedlings were grown in WL condition for 4 days and then treated with/without dark for 30 h. A, Mutant plants (phyB,pifq,myc2myc4,phyBmyc2myc4,pifqmyc2myc4) were grown in white light until 4-weeks old. Scale bar = 5 cm. B, Inflorescence stem length in various mutants. Student's t test (**P < 0.01) was used for statistical analysis, n > 10, mean ± SD. C, Transmission electron micrographs of stem cross sections showing interfascicular fiber cells. Scale bar = 5 μm. D, Statistics of SCW thickness in interfascicular fiber cells in (C). Data were collected from three biological replicates and more than 10 cells were measured per biological replicate. Student's t test (**P < 0.01) was used for statistical analysis, mean ± SD. E, F, Expression of the key SCW regulatory (NST1) and biosynthesis-related (4CL1 & IRX8) genes in mutant plants. Three biological replicates were performed. Student's t test (**P < 0.01, *P < 0.05) was used for statistical analysis, mean ± SD.

Figure 8 A proposed model of shaded light regulation of the SCW thickening.
White light enhances SCW thickening in fiber cells of inflorescence stem. Under white light (high R:FR condition), PHYB is activated to its Pfr form which enters the nucleus to inhibit PIF activity, MYC2 is available to bind to the NST1 promoter to activate the NST1-direct SCW thickening process. Under shaded light (low R:FR condition), PHYB reverts to its inactive Pr form. PHYB cannot enter the nucleus and PIF proteins interact with MYC2 and displaces its binding to the NST1 promoter. Thus, the NST1-directed SCW thickening process is suppressed.