Plant epidermal cell wall remodeling specific to meristem development and plant yield

Abstract

The present application relates to plant epidermis specific cell wall remodeling promotes meristem development and plant yield. Specifically, the present application provides a reagent for promoting plant epidermis specific expression of CSLD5 or CSLD4 protein or gene in promoting cell wall synthesis, improving the tissue mechanical properties of shoot apical meristem, and improving the traits of plants.

Description

Technical Field

[0001] This invention belongs to the field of botany, and more specifically, this invention relates to plant epidermal cell wall remodeling that promotes meristematic tissue development and plant yield. Background Technology

[0002] The development of tissues and organs in plants is a continuous process that spans the entire life cycle. This developmental characteristic is mainly attributed to the plant's ability to maintain stem cells throughout its lifespan. These stem cells, located in specific parts of the plant called meristems, provide new cells for organ development through asymmetric division, synthesis, and expansion of their cell walls.

[0003] Cell wall synthesis and expansion are fundamental to plant morphogenesis (Somerville et al., 2004). In the shoot apical meristem, the source of all aboveground tissues (Meyerowitz, 1997; Greb and Lohmann, 2016), biochemical and biomechanical signals converge at the cell wall, jointly regulating cell and organ morphology (Hamant et al., 2008; Coen and Cosgrove, 2023). Like peptides, hormones, and other signaling molecules, cell wall-mediated mechanomechanical processes are considered crucial for stem cell growth and organ differentiation.

[0004] Previous studies have shown that mutations in the cellulose synthase (CESA) gene can lead to defects in shoot apical meristem development (Sampathkumar et al., 2019), disruption of xyloglucan homeostasis can alter the geometry of meristem (Zhao et al., 2019), and increased pectin methyl esterification can inhibit the formation of floral primordia (Peaucelle et al., 2011).

[0005] Although cell wall remodeling has been extensively studied in non-dividing differentiated cells, such as Arabidopsis hypocotyl cells, the effects of cell wall synthesis on tissue mechanics and cell growth in proliferating stem cells remain largely unknown. Summary of the Invention

[0006] The purpose of this invention is to provide a specific cell wall remodeling method for plant epidermis that promotes meristematic tissue development and plant yield.

[0007] In a first aspect, the present invention provides an agent for promoting the specific expression of CSLD5 or CSLD4 protein or gene in plant epidermis, and its application in promoting cell wall synthesis, improving the tissue mechanical properties of shoot apical meristem, and improving plant traits.

[0008] In one or more embodiments, the cell wall synthesis includes: the synthesis of new cell walls, the expansion of interphase cell walls, the anisotropy of cell growth, the construction of cell plates during cell division, and the addition of new materials to existing cell walls during growth.

[0009] In one or more embodiments, the improved tissue mechanical properties of the shoot apical meristem include: enhanced cell wall stiffness and rigidity, and increased expression levels of mechanical stress-responsive genes.

[0010] In one or more embodiments, the enhancement of cell wall stiffness and rigidity includes an increase in the Young's modulus of the anticlinal wall of dividing and interphase cells.

[0011] In one or more embodiments, the mechanical stress response gene includes: TCH2, TCH3, TCH4, CML23, CML38, CPK28, CPK32, WRKY53, WRKY40, EFR11, PSK3, NHL3, AT1G76600, or HSPRO2.

[0012] In one or more embodiments, the improvement of the plant traits includes: promoting the growth of plant shoot apical meristem, increasing the size of plant shoot apical meristem, increasing plant weight, increasing plant leaf area, promoting the growth and development of shoot apical meristem cells, increasing the number of cells in L1 and L2 layers, increasing the number of plant divisions, promoting the increase of plant branches, increasing the number of tillers, increasing the number of primary branches and secondary branches per spike, increasing the number of grains per spike, and promoting the increase of plant yield.

[0013] In one or more embodiments, the plant epidermal-specific expression includes: specific expression in L1 layer cells of the plant, preferably specific expression in L1 layer cells of the plant shoot apical meristem.

[0014] In one or more embodiments, the method of plant epidermal-specific expression includes: using a plant epidermal-specific promoter to guide the specific expression of CSLD5 or CSLD4 protein or gene in the plant epidermis.

[0015] In one or more embodiments, recombinant DNA technology is used to insert a plant epidermal-specific promoter into an expression vector to control the expression of downstream CSLD5 or CSLD4 protein-coding sequences or genes.

[0016] In one or more embodiments, the plant epidermal-specific promoters include: ATML1 promoter, ROC1 promoter, GlymaML1 (LOC100782240) promoter, PtrML1 (LOC7458546) promoter, SIHDZIV7 (LOC7458546) promoter, TaROC2 (LOC123054665) promoter, and / or ZmHDZIV6 (LOC100279217) promoter.

[0017] In one or more embodiments, the reagent includes: a plant epidermal-specific promoter, a CSLD5 or CSLD4 protein-coding sequence or gene, a recombinant nucleic acid molecule, a vector, and a host cell.

[0018] In one or more embodiments, the plant epidermal-specific promoters include: ATML1 promoter, ROC1 promoter, GlymaML1 (LOC100782240) promoter, PtrML1 (LOC7458546) promoter, SIHDZIV7 (LOC7458546) promoter, TaROC2 (LOC123054665) promoter, and / or ZmHDZIV6 (LOC100279217) promoter.

[0019] In one or more embodiments, the recombinant nucleic acid molecule comprises: a plant epidermal-specific promoter, and a CSLD5 or CSLD4 protein-coding sequence or gene; preferably DNA or RNA.

[0020] In one or more embodiments, the vector includes an expression vector. In one or more embodiments, the expression vector includes a plasmid, an adenovirus vector, a lentivirus vector, or an adeno-associated virus vector.

[0021] In one or more embodiments, the plant is a plant containing shoot apical meristem. In one or more embodiments, the plant includes dicotyledonous plants and monocotyledonous plants. In one or more embodiments, the plant includes: grasses, cruciferous plants, and legumes. In one or more embodiments, the plant includes: Arabidopsis thaliana, rice, corn, wheat, barley, sorghum, rye, oats, sugarcane, soybean, alfalfa, rapeseed, sugar beet, tomato, cotton, sunflower, poplar, eucalyptus, and willow.

[0022] A second aspect of the present invention provides a method for promoting plant cell wall synthesis, improving the tissue mechanical properties of shoot apical meristems, and / or improving plant traits, the method comprising: specifically expressing CSLD5 protein or gene in the plant epidermis.

[0023] In one or more embodiments, the plant epidermal-specific expression is as described in any embodiment of the present invention.

[0024] In one or more embodiments, the promotion of plant cell wall synthesis, improvement of shoot apical meristem tissue mechanical properties, and / or improvement of plant traits are as described in any embodiment of the present invention.

[0025] In one or more embodiments, the plant is as described in any embodiment of the present invention.

[0026] A third aspect of the present invention provides a method for identifying plant cell wall synthesis capacity, shoot apical meristem tissue mechanical properties and / or traits, comprising: analyzing the expression level or activity of the CSLD5 or CSLD4 gene in the plant, and / or the expression level or activity of the CSLD5 or CSLD4 protein; if the expression level or activity of the CSLD5 or CSLD4 gene, and / or the expression level or activity of the CSLD5 or CSLD4 protein are normal, then the plant cell wall synthesis capacity, shoot apical meristem tissue mechanical properties and / or traits are normal; otherwise, the plant cell wall synthesis capacity, shoot apical meristem tissue mechanical properties and / or traits are abnormal.

[0027] In one or more embodiments, the promotion of plant cell wall synthesis, improvement of shoot apical meristem tissue mechanical properties, and / or improvement of plant traits are as described in any embodiment of the present invention.

[0028] In one or more embodiments, the plant is as described in any embodiment of the present invention.

[0029] A fourth aspect of the present invention provides a method for screening substances (potential substances) that regulate the ability of plant cell wall synthesis, the histomechanical properties and / or traits of shoot apical meristems, comprising:

[0030] (1) Add the candidate material to the system expressing CSLD5 or CSLD4;

[0031] (2) Detect the system and observe the expression or activity of CSLD5 or CSLD4. If the expression or activity is increased, it indicates that the candidate substance can be used to promote plant cell wall synthesis, shoot apical meristem tissue mechanical properties and / or improve plant traits; if the expression or activity is decreased, it indicates that the candidate substance can be used to inhibit plant cell wall synthesis, shoot apical meristem tissue mechanical properties and / or plant traits.

[0032] In one or more embodiments, the promotion of plant cell wall synthesis, improvement of shoot apical meristem tissue mechanical properties, and / or improvement of plant traits are as described in any embodiment of the present invention.

[0033] In one or more embodiments, the plant is as described in any embodiment of the present invention.

[0034] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0035] Figure 1 Mutations in glycosyltransferase genes affect the development of shoot apical meristems.

[0036] (a) GT mutants were used for shoot apical meristem (SAM) analysis. The left figure is a schematic diagram of cell wall tissue. The primary cell walls of meristem cells are mainly composed of cellulose, hemicellulose, pectin, and cell wall proteins, which are usually modified by carbohydrate side chains. Mutants were selected based on the mRNA expression pattern of each gene. The right figure shows the classification of 29 GT genes expressed in meristem: Type 1 indicates high and uniform expression, Type 2 indicates enrichment in floral primordia, Type 3 indicates expression only in dividing cells, Type 4 indicates widespread expression, but at a higher level in dividing cells, and Type 5 indicates expression in the procambium.

[0037] (b) Quantitative analysis of shoot apical meristem (SAM) size in GT mutants. SAM size was determined by measuring the average of three consecutive primitive radii to the SAM center. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (two-tailed t-test, compared with Col-0). NS, not significant.

[0038] Figure 2 CSLD5 regulates the growth and division of SAM cells.

[0039] The cells in the L1 layer were quantified using MorphoGraphX ​​software. The cell size (a), cell growth (b), main growth direction (c), and cell division (d) of wild-type (WT) and csld5 mutant (csld5) L1 layer SAM cells were analyzed. The left side is a schematic diagram, and the right side is a statistical graph. Figure 2 The scale bar in (a)-(d) is 50 μm.

[0040] Figure 3 CSLD5 regulates cell wall synthesis and cell wall mechanical stress.

[0041] (a) Comparison of cell wall morphology in wild-type (WT), csld5 mutant (csld5), and csld2csld5 double mutant (csld2csld5) SAM cells. Red lines mark newly formed cell walls within 12 hours (WT), 24 hours (csld5), or 72 hours (csld2csld5). New cell walls are indicated by arrows. Scale bar is 10 μm.

[0042] (b) Results of atomic force microscopy (AFM) observation of wild-type (WT) and csld5 mutant (csld5) SAM cells (left, scale bar is 20 μm) and statistical graph of Young's modulus of anticlinal wall of SAM dividing cells and interphase cells (right).

[0043] (c) Gene function annotation (GO) analysis of differentially expressed genes in stem meristems of WT and csld5. Notably, genes involved in DNA binding were downregulated in csld5, indicating a transcriptional response to cell wall defects.

[0044] (d) Analysis of stress genes regulated by CSLD5. The top figure is a Venn diagram showing the overlap between CSLD5-downregulated genes and the core transcriptome of mechanical stress in WT. Using gene-specific primers, qRT-PCR was used to verify the expression of representative stress genes in WT and CSLD5 shoot apex anatomical specimens. The bottom figure shows the classification of genes in the overlapping region.

[0045] (e) In tobacco (Nicotiana benthamiana) leaves, the interaction between GFP-labeled CSLD5 and FLAG-labeled CESA1, CESA3 or CESA6 was detected by immunoprecipitation using GFP-specific antibody (Anti-GFP) and FLAG-specific antibody (Anti-FLAG).

[0046] (f) Immunofluorescence images of the cellulose-binding module CBM4 in csld5 mutant (csld5) SAM cells (left, scale bar at 20 μm) and statistical graphs of CBM4 fluorescence intensity in wild-type (WT) and csld5 mutant (csld5) SAM cells [right, *P<0.05 (two-tailed t-test, compared with Col-0)].

[0047] Figure 4 MYB3R4 activates the expression of CSLD5 in dividing cells.

[0048] (a) Schematic diagram of the CSLD5 genomic region, with colored circles indicating the locations of MSA elements. “I” and “II” indicate regions analyzed by ChIP PCR.

[0049] (b) Chromatin immunoprecipitation sequencing (ChIP-seq) verified that the five MSA elements could be recognized by the MYB3R transcription factor.

[0050] (c)ChIP real-time quantitative PCR (qPCR) to verify the binding of MYB3R1 and MYB3R4 to the CSLD5 promoter.

[0051] (de) Gel migration assay (EMSA) showed that MBP-MYB3R1 and MBP-MYB3R4 could bind to Cy5-labeled DNA probes, but MBP could not bind to Cy5-labeled DNA probes alone.

[0052] (f) Dual-luciferase assays showed transcriptional activation of CSLD5 by MYB3R1 and MYB3R4. The ratio of LUC to REN activity is shown in tobacco leaf cells co-expressing the pCSLD5::LUC reporter gene with MYB3R1 or MYB3R4. REN expression is driven by the 35S promoter. Data are from 6 biological replicates; error bars represent mean ± SEM.

[0053] (gh)(g) qRT-PCR was used to compare the transcriptional levels of CSLD5 in WT and myb3r1 myb3r4 SAMs. (h) RNA FISH was used to compare the expression of CSLD5 mRNA in WT and myb3r1 myb3r4 SAMs. CSLD5 mRNA probes were labeled with DIG and detected by TSA-CY5 (red signal). Cell nuclei were stained with DAPI, appearing blue. FISH signals are also displayed in Fire format. The right figure shows the quantification of RNA FISH fluorescence intensity. The scale bar is 50 μm. The results of qRT-PCR (g) and RNA FISH (h) experiments showed that the expression level of CSLD5 was significantly reduced in the myb3r1 myb3r4 double mutant.

[0054] (i) Expression map of pCSLD5::GFP-CSLD5 at SAM. GFP-CSLD5 protein is localized in cytoplasm and developing cell plate. Scale bars are 20 μm (four images on the left) and 5 μm (two images on the right).

[0055] (j) Time-lapse imaging of GFP-CSLD5 during cell division. The plasma membrane was labeled with 29-1-tdTomato. GFP-CSLD5 localized to the cell plate before the 29-1-tdTomato signal was observed. The fluorescence signals of GFP-CSLD5 and 29-1-tdTomato along the line shown in the lower left corner were quantitatively measured. The scale bar is 5 μm. The right figure shows the fluorescence intensity statistics.

[0056] (k)GFP-CSLD5 partially co-localizes with VHA-RFP, a marker of the trans-Golgi network (TGN) and early endosomes. Scale bar: 5 μm.

[0057] Figure 5 CSLD5-mediated cell wall synthesis feeds back into the cell cycle process.

[0058] (ab) RNA FISH comparison of HIS4 (a) and CYCB1;2 (b) mRNA expression in wild-type and csld5 SAMs. Immunofluorescence assays showed that compared to wild-type (WT), the expression level of HIS4 mRNA in csld5 mutant (csld5) SAM cells was significantly reduced (a), while the expression level of CYCB1;2 mRNA was increased (b). HISTONE 4 (HIS4) mRNA probes were labeled with DIG and detected using TSA-CY5 (red signal). CYCLIN B1;2 (CYCB1;2) mRNA probes were labeled with fluorescein isothiocyanate (FITC) and detected using TSA-FITC (green signal). Cell nuclei were stained with DAPI, appearing blue. FISH signals are displayed in Fire format. The right figure shows the quantification of RNA FISH fluorescence intensity. Scale bar is 50 μm.

[0059] (c) Schematic diagram of the cell cycle during mitosis. HIS4 and CYCB1;2 are specifically expressed during the G1-S and G2-M transition phases, respectively. Cell plate formation occurs at telophase of mitosis.

[0060] (d) A schematic diagram showing how mutations in CSLD5 lead to cell capture during the early stages of mitosis before cell plate formation.

[0061] Figure 6 Functional analysis of CSLD2, CSLD3 and CSLD5 in plant growth and SAM development regulation.

[0062] (a) Yeast two-hybrid experiments showed that CSLD5 and CSLD3 interact. The interaction between CSLD5 and CSLD3 was studied using a cleavage-ubiquitin membrane yeast two-hybrid system. CSLD5 fused with the Nub fragment encoded by pXGY18, and CSLD3 fused with the Cub fragment encoded by pXGY17.

[0063] (b) Regulation of GFP-CSLD2 and GFP-CSLD3 expression patterns by the CSLD5 promoter. GFP-CSLD2 and GFP-CSLD3 are highly expressed in actively proliferating cells under the CSLD5 promoter. Scale bar: 50 μm.

[0064] (c) Both GFP-CSLD2 and GFP-CSLD3 proteins are localized to the cell plate and exhibit subcellular dynamics similar to CSLD5. Scale bar: 5 μm.

[0065] (dg) Growth phenotype (de) and three-dimensional projection (fg) of SAMs. Cell walls were stained with propidium iodide (PI) and shown as grayscale. Quantitative analysis of plant weight (e) and SAM size (g) showed that the expression of GFP-CSLD2, GFP-CSLD3, and GFP-CSLD5 completely restored the phenotype of the csld5 mutant under the control of the CSLD5 promoter. Scale bars are 2 cm (d) and 50 μm (f).

[0066] Figure 7 CSLD5 interacts with and co-localizes with primary cell wall CESAs.

[0067] (a) Yeast two-hybrid assays showed a strong interaction between CSLD5 and CESA3 or CESA6, and a weaker interaction between CSLD5 and CESA1. CSLD5 fused with the Nub fragment encoded by pXGY18, and all three CESAs fused with the Cub fragment encoded by pXGY17.

[0068] (b) Co-localization of CSLD5 and CESA proteins. mCherry-CSLD5 and GFP-CESAs were transiently co-expressed in tobacco cells. Scale bar: 5 μm.

[0069] (ce) The subcellular localization of CSLD5 and CESA proteins in Arabidopsis root cells is achieved through hybridization of pCSLD5::mCherry-CSLD5 with pUBQ10::GFP-CESA1, pUBQ10::GFP-CESA3, or pUBQ10::GFP-CESA6. Immunofluorescence assays showed that in stable transgenic plants co-expressing mCherry-CSLD5 with GFP-CESA1 (c), GFP-CESA3 (d), or GFP-CESA6 (e), CSLD5 and CESA3 co-modify the cell plate, and co-localization of CSLD5 with CESA1 or CESA6 on the cell plate is also evident. Extensive overlap between CSLD5 and CESA proteins was also observed in the putative Golgi apparatus stack. The upper part of the figure shows interphase cells, and the lower part shows cells in anaphase of mitosis, at which point the cell plate (indicated by the arrow) is forming. The scale bar is 5 μm.

[0070] Figure 8 CESA3 reverted to the csld5 mutant phenotype.

[0071] (a) Using a two-way genetic complementation experiment, CESA1, CESA3, and CESA6, GFP-tagged cells driven by the CSLD5 promoter, were expressed in the csld5 mutant. Wild-type (WT) and csld5 mutant plants (csld5) were used as controls. The results showed that CESA1 had little effect on the growth of csld5, while csld5;pCSLD5::GFP-CESA6 plants exhibited severe growth retardation. Conversely, GFP-CESA3 was found to completely rescue the growth defects of csld5. The top image shows the plant phenotype, and the bottom image shows the 3D projection of SAMs. Cell walls were stained with propidium iodide (PI) and displayed in grayscale. Scale bars are 2 cm (top image) and 50 μm (bottom image).

[0072] (b) Quantify the weight of wild-type, csld5, and csld5 plants expressing GFP-CESA1, GFP-CESA3, or GFP-CESA6.

[0073] (c) Quantification of SAM size in wild-type, csld5, and csld5 plants expressing GFP-CESA1, GFP-CESA3, or GFP-CESA6.

[0074] Figure 9 The widely expressed GFP-CSLD5 partially reverted to the csld5 mutant phenotype.

[0075] (a) Expression pattern of pRPS5A::GFP-CSLD5 in SAM. Cell walls stained magenta with propidium iodide (PI). Immunofluorescence assays showed that the RPS5A promoter-driven GFP-CSLD5 protein was expressed throughout the SAM. Scale bar: 50 μm.

[0076] (b) Side view of a SAM expressing pRPS5A::GFP-CSLD5. The cell wall was stained magenta with propidium iodide (PI). The image below is a magnified view of the area within the white box in the image above. Scale bars are 50 μm (top part) and 5 μm (bottom part).

[0077] (c) Comparison of SAM morphology in wild-type, csld5, and csld5 plants expressing pRPS5A::GFP-CSLD5. Scale bar: 50 μm.

[0078] (df) Quantitative analysis of SAM size (d), plant weight (e), and leaf area (f) of wild-type, csld5, and csld5 plants.

[0079] Figure 10 L1 expression of CSLD5 completely reverted to the CSLD5 mutant phenotype.

[0080] (ab) RNA fluorescence in situ hybridization (FISH) showed that GFP-CSLD5 mRNA was specifically detected only in L1 cells of SAM. (b) is a magnified view of the white box area in (a), with L1, L2, and L3 indicating the locations of L1, L2, and L3 cells in SAM, respectively. GFP-CSLD5 chimeric mRNA was detected using a GFP-specific probe. Cell nuclei were stained blue with DAPI. Scale bars are 50 μm (a) and 10 μm (b).

[0081] (c) Comparison of SAM morphology in WT, csld5, and csld5; pATML1::GFP-CSLD5 plants. pATML::GFP-CSLD5 was transformed into csld5 mutant plants, with wild-type (WT) and csld5 mutant plants used as controls. The results showed that GFP-CSLD5 expressed in the L1 layer completely rescued the SAM and plant size of csld5. Scale bar: 50 μm.

[0082] (d) Morphology of WT, csld5, and csld5; pATML1:GFP-CSLD5 plants at bolting stage. Scale bar is 2 cm.

[0083] Statistical plots of SAM size and leaf size for (ef)WT, csld5, and csld5;pATML::GFP-CSLD5. Compared with csld5 and wild-type plants, csld5;pATML::GFP-CSLD5 showed significantly increased SAM size and leaf size.

[0084] Figure 11 GFP-CSLD5 expressed at SAM by L1 restores inner layer growth and stress transcriptome.

[0085] (ab) Confocal microscopy analysis showed that the GFP-CSLD5 protein was specifically detected only in L1 cells of SAM. (a) The top layer of a SAM expressing pATML1::GFP-CSLD5 is shown in the image. The cell wall is stained with propidium iodide (PI) and appears magenta. The scale bar is 50 μm. (b) The image is a side view of SAMs, showing the specific expression of pATML1::GFP-CSLD5 in L1 cells. The right image is a magnified view of the white box in the left image. L1, L2, and L3 indicate the positions of L1, L2, and L3 cells in the SAM, respectively. The cell walls are stained with propidium iodide (PI) and appear magenta. The scale bars are 50 μm (left) and 10 μm (right).

[0086] Quantitative analysis of cell size in L1(c) and L2(d) layers of SAMs from (cd) WT, csld5, and csld5; pATML1::GFP-CSLD5 plants. Cells were segmented in MorphoGraphX ​​and displayed as a heatmap (top). The projection of cells is shown in the bottom image, with a scale bar of 20 μm.

[0087] (ef) Statistical graph of L1 cells (e) and L2 cells (f) of SAMs with wild type (WT), csld5 mutant, csld5; pATML::GFP-CSLD5.

[0088] (g) Hierarchical clustering analysis of 117 CSLD5-related tactile genes in (g)WT, csld5, and pATML1::GFP-CSLD5 SAMs. RNA sequencing analysis showed that 79.5% (93 / 117) of CSLD5 maintenance and mechanical stress response genes reverted to wild-type levels through CSLD5 expression in the L1 layer. After epidermal GFP-CSLD5 treatment, the expression of most genes recovered to wild-type levels.

[0089] Figure 12 CSLD5 expressed by L1 restores the expression of cell wall mechanical, transcriptomic, and mechanical stress response genes.

[0090] (a) Hierarchical clustering of differentially expressed genes in WT, csld5, and csld5;pATML1::GFP-CSLD5 SAMs. RNA sequencing analysis showed that transcriptomic changes in csld5 SAMs were also restored in csld5;pATML::GFP-CSLD5 plants.

[0091] (b) Relative expression levels of TCH2, TCH3, TCH4, CML23, CML38, CPK28, CPK32, WRKY53, WRKY40, EFR11, PSK3, NHL3, AT1G76600 and HSPRO2 genes in wild-type (WT), csld5 mutant, csld5; pATML::GFP-CSLD5 plants.

[0092] (c) Atomic force microscopy measurements show that csld5;pATML::GFP-CSLD5 SAM is harder than csld5 alone (Cohen's d = 0.839476). The left image shows the atomic force microscopy measurements, with a scale bar of 20 μm. The right image shows the statistical plot of Young's modulus.

[0093] Figure 13 OsCSLD4 expressed in the epidermis promotes the growth of rice meristems.

[0094] (a) GFP-OsCSLD4 was expressed in the wild-type background of Zhonghua 11 (ZH11) under the control of the epidermal-specific promoter ROC1. RNA FISH assay confirmed that the mRNA of GFP-OsCSLD4 was confined to epidermal cells. +DAPI is used to show the cell nucleus, which appears in blue. Scale bar is 50 μm.

[0095] (b) Comparison of HISTONE4 (OsHIS4, Os10g0539500) mRNA expression in stem meristems of wild-type (ZH11) and pROC1::GFP-OsCSLD4 plants. GFP-OsCSLD4 was expressed in wild-type Zhonghua 11 (ZH11) rice under the control of the epidermal-specific promoter ROC1. The results showed that the shoot apical meristems of pROC1::GFP-OsCSLD4 plants were larger than those of wild-type plants and exhibited higher levels of HIS4 expression. Scale bar: 150 μm.

[0096] (cf) Statistical graphs of branch length (c), plant height (d), spike length (e), and thousand-grain weight (f) of pROC1::GFP-OsCSLD4 transgenic plants, with the wild-type Zhonghua 11 (ZH11) as a control. These agronomic traits were not affected by epidermal expression of OsCSLD4. ns, not significant.

[0097] Figure 14 Epidermal cell wall synthesis promotes rice seedling growth.

[0098] (a) GFP-OsCSLD4 was expressed in the wild-type background of Zhonghua 11 (ZH11) under the control of the epidermal-specific promoter ROC1. Confocal imaging confirmed that the GFP-OsCSLD4 protein was confined to epidermal cells. Scale bar: 100 μm.

[0099] (bc) Phenotypic (b) and branching (c) images of wild-type Zhonghua 11 (ZH11) plants and two pROC1::GFP-OsCSLD4 transgenic plants. The pROC1::GFP-OsCSLD4 plants produced more tillers than ZH11. Expression of GFP-OsCSLD4 in L1 cells promoted spike growth. The pROC1::GFP-OsCSLD4 gene resulted in increased spike branching, especially secondary branching. Scale bars are 10cm (b) and 3cm (c).

[0100] (df) Statistics on the number of divisions (d), number of branches (e, the left figure is the statistics of the number of first-order branches, and the right figure is the statistics of the number of second-order branches) and number of grains per ear of wild-type plants (ZH11) and transgenic plants (pROC1::GFP-OsCSLD4). Detailed Implementation

[0101] Through in-depth research, the inventors discovered that CSLD5-mediated cell wall synthesis regulates tissue mechanics and intercellular growth balance in the shoot apical meristem (SAM) of Arabidopsis thaliana. The Myb family transcription factor MYB3R4 directly activates CSLD5 expression, promoting robust synthesis of new cell walls in dividing cells. CSLD5 co-localizes with key cellulose synthases CESAs (including CESA1, CESA3, and CESA6) responsible for primary cell wall synthesis, forming a complex that guides cellulose-based cell wall construction. Impaired CSLD5 function leads to slow cell growth, decreased cell wall stiffness, and altered expression of mechanical stress-responsive genes. Confining CSLD5 to L1 cells restores the mechanical properties and growth defects of the CSLD5 mutant SAM, and this salvage effect extends to L2 cells, indicating the existence of molecular and cellular compensation mechanisms between cell layers in the shoot apical meristem. Further research shows that specific expression of OsCSLD4 in the rice epidermis enhances inflorescence meristem growth and seed yield. Therefore, by using biological methods to specifically express CSLD5 or CSLD4 in the plant epidermis, the development of shoot tip meristem can be promoted through the interlayer coordination mechanism mediated by cell wall synthesis, thereby increasing plant yield. This method can be applied to improve plant varieties and cultivate high-yielding plants.

[0102] As used herein, "plant" generally refers to a plant possessing stem apical meristem (SAM), and may be (but is not limited to): dicotyledonous plants and monocotyledonous plants. More specifically, "plant" includes, but is not limited to: grasses, cruciferous plants, legumes, etc. Preferably, the plant is a grass or a cruciferous plant. For example, the "plant" includes, but is not limited to: Arabidopsis thaliana, rice, corn, wheat, barley, sorghum, rye, oats, sugarcane, soybean, alfalfa, rapeseed, sugar beet, cotton, sunflower, poplar, eucalyptus, willow, etc. Preferably, the "plant" includes, but is not limited to: rice, Arabidopsis thaliana, etc.

[0103] As used herein, "shoot apical meristem" or "SAM" generally refers to the tissue located at the apex of a plant that has the ability to continuously differentiate. It can produce organs such as stems, branches, leaves, and flowers through cell division and differentiation, forming the above-ground parts of the plant. SAM can usually be divided into three layers: L1, L2, and L3.

[0104] As used herein, the terms “increase,” “enhance,” “improve,” or “enhance” are interchangeable and, in their application, should mean an improvement of at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35%, 40%, or more, in plant traits compared to a control plant as defined herein. Regarding “control plant,” selecting a suitable control plant is a routine part of the experimental design and may include a corresponding wild-type plant, a plant with a growth defect, etc. Control plants are generally the same plant species or even the same variety as the plant being evaluated. Control plants may also be plants that have partially or completely lost the function of the target gene due to mutation. As used herein, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0105] The functions of CSLD5 or CSLD4, and their relationship with cell wall synthesis, tissue mechanics, and cell growth in shoot apical meristem, are not well understood in this field. In this invention, through genetic screening, the inventors discovered that CSLD5 is expressed only in dividing cells and is a key regulator of shoot apical meristem growth. Further research revealed that CSLD5 mediates the synthesis and expansion of newly formed cell walls, maintains the mechanical integrity of cell walls, influences cell cycle progression, and exhibits strong interactions with CESA3 or CESA6. Impaired CSLD5 function leads to slow cell growth, decreased cell wall stiffness, and altered expression of mechanical stress response genes. CSLD5 specifically expressed in plant epidermis can restore the mechanical properties and growth defects of csld5 mutant shoot apical meristem. Further research shows that specific expression of OsCSLD4 in rice epidermis enhances inflorescence meristem growth and seed yield.

[0106] As used herein, the term "CSLD" refers to a family of cellulose synthase-like enzymes that belongs to the Cellulose Synthase-Like (CSL) superfamily, and has the highest similarity to CESAs (Richmond and Somerville, 2000). CSLD5 (Cellulose Synthase Like-D5) is a subtype of CSLD, and the amino acid sequence of the CSLD5 protein in Arabidopsis thaliana is shown in SEQ ID NO:1. Any protein / gene that shares high homology (e.g., 80% or higher; preferably 90% or higher, such as 95%, 98%, or 99%) with the CSLD5 specifically referenced in this invention and has the same function as said CSLD5 is also included in this invention. CSLD4 (Cellulose Synthase Like-D4) is another subtype of CSLD, and the amino acid sequence of the CSLD4 protein in rice (Oryza sativa) is shown in SEQ ID NO:2. Any protein / gene that shares high homology (e.g., 80% or higher; preferably 90% or higher, such as 95%, 98% or 99%) with the CSLD4 specifically referenced in this invention and has the same function as the CSLD4 is also included in this invention.

[0107] In this invention, CSLD5 or CSLD4 also includes its homologs, namely proteins / genes that exist in species other than Arabidopsis thaliana and rice, have homology with CSLD5 or CSLD4 and have the same function as CSLD5 or CSLD4 in this invention, or play the same or similar role in the same or similar signaling pathways. Since CSLD5 or CSLD4 is conserved in multiple species, it should be understood that although the CSLD5 or CSLD4 proteins / genes presented in the embodiments of this invention are preferred, this invention is not limited to the proteins / genes specifically listed in the embodiments.

[0108] This invention provides reagents for promoting the specific expression of CSLD5 or CSLD4 protein / genes in plant epidermis, and their application in promoting cell wall synthesis, improving the mechanical properties of shoot apical meristems, and enhancing plant traits. Related applications include plant epidermal-specific promoters, vectors containing the promoter and the CSLD5 or CSLD4 gene, host cells transferred into the vector, transgenic plant cells, tissues, or other explants and their preparation methods, methods for preparing transgenic plants, and transgenic plants, etc.

[0109] The vectors include expression vectors, such as, but not limited to, plasmids, adenovirus vectors, lentivirus vectors, adeno-associated virus vectors, etc.

[0110] The method for preparing the transgenic plant cells, tissues or other explants includes: introducing an expression vector containing a plant epidermal-specific promoter and a coding sequence for CSLD5 or CSLD4 protein into plant cells, tissues or other explants for expression, thereby obtaining plant cells, tissues or other explants in which CSLD5 or CSLD4 is specifically expressed in the plant epidermis (e.g., L1 layer cells of shoot apical meristem).

[0111] Plant transformation using recombinant DNA can be achieved through methods such as Agrobacterium-mediated transformation or gene gun transformation, including leaf disc transformation and rice embryo transformation. Specifically, the method for preparing transgenic plants includes: introducing an expression vector containing a plant epidermal-specific promoter and a coding sequence for CSLD5 or CSLD4 protein into plant cells, tissues, or other explants for expression; the CSLD5 protein in the plant cells, tissues, or other explants is specifically expressed in the plant epidermis (e.g., L1 layer cells of the shoot apex meristem); and then regenerating the plant cells, tissues, or other explants into plants. The transgenic plant also includes plant parts, such as leaves, stems, roots, tubers, flowers, seeds, kernels, grains, and fruits.

[0112] As a preferred embodiment of the present invention, the method for preparing trait-improved transgenic plants is as follows:

[0113] (1) An expression vector is provided, wherein the expression vector contains a plant epidermal-specific promoter and a coding sequence for CSLD5 or CSLD4 protein;

[0114] (2) The expression vector from step (1) is transferred into plant epidermal tissues or organs, thereby enabling the coding sequences of CSLD5 or CSLD4 proteins to be specifically expressed in plant epidermal tissues or organs.

[0115] (3) Regenerate plants from the plant epidermal tissues or organs that specifically express CSLD5 or CSLD4 protein or gene obtained in step (2).

[0116] The present invention also provides a method for promoting plant cell wall synthesis, improving the tissue mechanical properties of shoot apical meristem and / or improving plant traits, comprising: specifically expressing CSLD5 or CSLD4 protein or gene in the plant epidermis.

[0117] In this invention, the plant epidermal-specific expression includes L1 layer cell-specific expression. As one embodiment of this invention, a plant epidermal-specific promoter can be used to guide the specific expression of CSLD5 or CSLD4 protein / gene in the plant epidermis (e.g., L1 layer cells of the shoot apex meristem). For example, recombinant DNA technology can be used to insert the plant epidermal-specific promoter into an expression vector to control the expression of the downstream CSLD5 or CSLD4 gene. Exemplary plant epidermal-specific promoters include, but are not limited to: ATML1 promoter, ROC1 promoter, GlymaML1 (LOC100782240) promoter, PtrML1 (LOC7458546) promoter, SIHDZIV7 (LOC7458546) promoter, TaROC2 (LOC123054665) promoter, and / or ZmHDZIV6 (LOC100279217) promoter.

[0118] By specifically expressing CSLD5 or CSLD4 protein / gene in plant epidermis (e.g., L1 layer cells of shoot apical meristem), a vector, host cell, or plant that specifically expresses CSLD5 or CSLD4 protein / gene in plant epidermis can be obtained. Compared with the wild type, it promotes cell wall synthesis, improves the tissue mechanical properties of shoot apical meristem, and improves plant traits.

[0119] The cell wall synthesis includes, but is not limited to: the synthesis of new cell walls, the expansion of interphase cell walls, the anisotropy of cell growth, the construction of cell plates during cell division, and the addition of new materials to existing cell walls during growth.

[0120] The improvement in the tissue mechanical properties of the shoot apical meristem includes, but is not limited to: increased cell wall stiffness and rigidity, and increased expression levels of mechanical stress-responsive genes; preferably, the increased cell wall stiffness and rigidity includes: increased Young's modulus of the anticlinal wall of dividing cells and interphase cells. The mechanical stress-responsive genes include, but are not limited to: TCH2, TCH3, TCH4, CML23, CML38, CPK28, CPK32, WRKY53, WRKY40, EFR11, PSK3, NHL3, AT1G76600, or HSPRO2.

[0121] The improvement of the plant traits includes, but is not limited to: promoting the growth of plant shoot tip meristem, increasing the size of plant shoot tip meristem, increasing plant weight, increasing plant leaf area, promoting the growth and development of shoot tip meristem cells, increasing the number of cells in L1 and L2 layers, increasing the number of plant divisions, promoting the increase of plant branches, increasing the number of tillers, increasing the number of primary branches and secondary branches per spike, increasing the number of grains per spike, and promoting the increase of plant yield.

[0122] The method described according to the present invention can be advantageously combined with other methods that promote the growth of plant shoot apical meristems, promote the synthesis and expansion of cell walls, enhance the tissue mechanical properties of shoot apical meristems, increase plant yield, or improve other plant characteristics.

[0123] Based on the inventors' new discovery, CSLD5 or CSLD4 can also be used as molecular markers for selecting superior plant varieties. By identifying the plants to be tested (such as seedlings or seeds) and analyzing the expression of CSLD5 or CSLD4 proteins or genes, a high expression level of CSLD5 or CSLD4 proteins or genes indicates that the plant has excellent cell wall synthesis ability, shoot apical meristem tissue mechanical properties, and / or superior traits; conversely, a low expression level indicates that the plant has defects in cell wall synthesis ability, shoot apical meristem tissue mechanical properties, and / or traits.

[0124] Therefore, the present invention also provides a method for identifying plant cell wall synthesis capacity, shoot apical meristem tissue mechanical properties and / or traits, comprising: analyzing the expression level or activity of the CSLD5 or CSLD4 gene in the plant, and / or the expression level or activity of the CSLD5 or CSLD4 protein; if the expression level or activity of the CSLD5 or CSLD4 gene, and / or the expression level or activity of the CSLD5 or CSLD4 protein are normal, then the plant cell wall synthesis capacity, shoot apical meristem tissue mechanical properties and / or traits are normal; otherwise, the plant cell wall synthesis capacity, shoot apical meristem tissue mechanical properties and / or traits are abnormal.

[0125] This invention also provides a method for screening substances (potential substances) that regulate plant cell wall synthesis capacity, shoot apical meristem tissue mechanical properties and / or traits, comprising:

[0126] (1) Add the candidate material to the system expressing CSLD5 or CSLD4;

[0127] (2) Detect the system and observe the expression or activity of CSLD5 or CSLD4. If the expression or activity is increased, it indicates that the candidate substance can be used to promote plant cell wall synthesis, shoot apical meristem tissue mechanical properties and / or improve plant traits; if the expression or activity is decreased, it indicates that the candidate substance can be used to inhibit plant cell wall synthesis, shoot apical meristem tissue mechanical properties and / or plant traits.

[0128] Methods for screening substances that act on proteins or genes as targets are well known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art will understand how to select an appropriate screening method.

[0129] Through large-scale screening, a class of potential substances with regulatory effects that specifically enhance the expression or activity of CSLD5 or CSLD4 can be obtained.

[0130] The advantages of this invention include:

[0131] (1) This invention is the first to discover that CSLD5 mediates the synthesis and expansion of new cell walls, maintains the mechanical integrity of cell walls, affects the cell cycle process, and has a strong interaction with CESA3 or CESA6.

[0132] (2) This invention discovered that impaired CSLD5 function leads to slow cell growth, decreased cell wall stiffness, and altered expression of mechanical stress response genes. By using biological methods to specifically express CSLD5 in the plant epidermis, the development of shoot apical meristem can be promoted through a cell wall synthesis-mediated interlayer coordination mechanism. Further research showed that specific expression of OsCSLD4 in the rice epidermis enhances inflorescence meristem growth and seed yield. Therefore, specifically regulating the expression or activity of CSLD5 or CSLD4 in the plant epidermis can increase plant yield and can be applied to improve plant varieties and cultivate high-yielding plants.

[0133] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0134] Materials and Methods

[0135] 1. Plant materials and growing conditions

[0136] This study used the Arabidopsis thaliana Columbia ecotype (Col-0) as a wild-type control. Mutants csld2, csld5, ixr1-2, and myb3r1 and myb3r4 were described in previous studies (Scheible et al., 2001; Yang et al., 2016, 2021). The csld2 and csld5 double mutants were obtained through hybridization. All GT mutants were ordered from the Nottingham Arabidopsis Germplasm Resource Centre. Seeds were surface-sterilized for 5 minutes with a mixture of 70% ethanol and 0.5% Triton X-100, followed by a brief rinse with 95% ethanol for 1 minute. Seeds were then dried under a sterile hood and germinated on horizontal or vertical plates containing semi-strength Murashige-Skoog medium. After 11 days, seedlings were transplanted into soil. The plants were cultured in the growth chamber under the following conditions: long day cycle (16 hours light / 8 hours dark), light intensity of 170 μmol / m² / s, and day / night temperatures of 21℃ / 17℃.

[0137] Rice (Oryza sativa) plants were grown in protected paddy fields in Shanghai or Hainan Island. Tobacco (Nicotianabenthamiana) plants were grown at 22°C under long-day conditions (16 hours of daylight / 8 hours of darkness), and leaves from 3- to 4-week-old plants were used for transient expression experiments.

[0138] 2. Plasmid construction and plant transformation

[0139] To construct pCSLD5::GFP-CSLD5, primers D5P-F (KpnI) and D5P-R (PstI) were used to amplify the 2,995 bp promoter sequence upstream of the CSLD5 start codon, primers GFP-F (PstI) and GFP-R (SpeI) were used to amplify the 717 bp GFP coding region, and primers D5DNA_F (SpeI) and D5DNA_R (XbaI) were used to amplify a 4,270 bp genomic fragment containing the full-length CSLD5 coding sequence and a 487 bp 3' end. These three fragments were sequentially cloned into the pBluescript SK-vector, forming SK-D5pro, SK-D5pro-GFP, and SK-D5pro-GFP-D5. The complete fragment containing the CSLD5 promoter, GFP, and CSLD5 coding region was digested with KpnI and XbaI and ligated into the pCAMBIA1300 vector.

[0140] To construct pCSLD5::mCherry-CSLD5, primers D5pro-F (EcoRI) and D5pro-R (KpnI) were used to amplify the promoter sequence 3,000 bp upstream of the start codon of CSLD5, and primers D5ter1k_F (XbaI) and D5ter1k_R (HindIII) were used to amplify the 3' end region 1,000 bp. The pCAMBIA1300 vector was digested with EcoRI and HindIII, and the two PCR products were inserted into the vector via homologous recombination (using ClonExpress, Vazyme) to obtain 1300-pCSLD5-MCS-CSLD5. ter1k A 708 bp mCherry coding region was amplified using primers mCherry-F (KpnI) and mCherry-R (KpnI), and a 3,777 bp genomic fragment containing the full-length CSLD5 coding sequence was amplified using primers D5g_F (KpnI) and D5g_R (BamHI). 1300-pCSLD5-MCS-CSLD5 ter1k After being digested with KpnI and BamHI, the vector was inserted into the mCherry and CSLD5 fragments via homologous recombination to generate pCSLD5::mCherry-CSLD5.

[0141] To construct pATML1::GFP-CSLD5, using 1300-pCSLD5::GFP-CSLD5 as a template, the GFP-CSLD5 fusion fragment was amplified using primers GFP-1300-SacI-F and CSLD5-R (PstI). The PCR product was then inserted into the 1300-Nos vector via homologous recombination to obtain 1300-GFP-CSLD5-Nos. Next, using genomic DNA as a template, the ATML1 promoter was amplified using primers pATML1-F and pATML1-R, and the ATML1 promoter was inserted into 1300-GFP-CSLD5-Nos via homologous recombination to form pATML1::GFP-CSLD5.

[0142] To construct pRPS5A::GFP-CSLD5, primers pRPS5A-F and pRPS5A-R were used to amplify the RPS5A promoter, and primers eGFP-F and eGFP-R were used to amplify the eGFP coding region. These two PCR products were inserted into the 1300-Nos vector via homologous recombination to generate 1300-pRPS5A-eGFP-MCS-Nos. The genomic fragment containing the full-length coding sequence of CSLD5 was amplified using primers CSLD5-F (SalI) and CSLD5-R (PstI), and then ligated into 1300-pRPS5A-eGFP-MCS-Nos via homologous recombination to obtain pRPS5A::GFP-CSLD5.

[0143] When constructing pCSLD5::GFP-CESA1, pCSLD5::GFP-CESA3, and pCSLD5::GFP-CESA6, the eGFP coding region was first amplified using primers eGFP-F / eGFP-R (BamHI), and then inserted into 1300-pCSLD5-MCS-CSLD5 via homologous recombination. ter1k The vector forms 1300-pCSLD5-eGFP-MCS-CSLD5 ter1k Subsequently, genomic fragments containing the full-length coding sequences of CESA1, CESA3, or CESA6 were amplified using primers CESA1-F(BamHI) / CESA1-R(PstI), CESA3-F(BamHI) / CESA3-R(PstI), and CESA6-F(BamHI) / CESA6-R(PstI), and then inserted into the aforementioned vectors via homologous recombination.

[0144] When constructing pUBQ10::GFP-CESA1, pUBQ10::GFP-CESA3, and pUBQ10::GFP-CESA6, primers CESA1-F(KpnI) / CESA1-R(BamHI), CESA3-F(KpnI) / CESA3-R(BamHI), and CESA6-F(KpnI) / CESA6-R(BamHI) were used to amplify genomic DNA fragments containing the full-length coding sequences of CESA1, CESA3, and CESA6, respectively. After digestion with KpnI and BamHI, the pCAMBIA1300-UBQ10-eGFP(N)-NOS vector was inserted into the corresponding PCR products via homologous recombination.

[0145] 3. Confocal microscopy imaging and data analysis

[0146] A. Preparation of Shoot Apical Meristem (SAM) Samples

[0147] During the bolting stage, the size, gene expression patterns, and subcellular protein dynamics of the shoot apex meristem (SAM) were analyzed. A 1 cm section of the shoot apex was excised, and the SAM was meticulously dissected under a stereomicroscope. After removing larger floral organs, the SAM was transferred to a container containing fresh MS medium (Murashige and Skog medium containing vitamins). To visualize cell boundaries, the SAM was stained with 0.1% propidium iodide (PI) for 2 minutes to mark the cell walls. The staining solution was rinsed off with sterile water before imaging.

[0148] B. Long-duration time-lapse imaging

[0149] To track cell division in SAM, plants were removed from the soil and transferred to covered boxes containing room-temperature Arabidopsis thaliana medium. Larger flowers were removed to expose the apical meristem. After 12 hours of recovery in a growth chamber with a 16 / 8-hour light / dark cycle, plants were imaged using confocal microscopy. Confocal Z-axis stacked images of SAM were acquired using a 25×NA 0.95 long working distance water immersion objective on a Leica SP8 microscope or a 40×NA 1.2 long working distance water immersion objective on a Zeiss LSM880 microscope. Laser excitation wavelengths were 488 nm (for PI and GFP) and 555 nm or 561 nm (for RFP and FM4-64). Three-dimensional reconstruction was performed in Fiji software.

[0150] C. In vivo imaging of rice apical meristem

[0151] During the transition from reproductive to primordial stage (approximately 60 days after sowing), the apex of the rice plant was dissected. Leaves and leaf sheaths were carefully removed to expose the flower primordia for confocal imaging.

[0152] D. Image Processing and Analysis

[0153] Cell size determination, cell growth, and cell line tracking were performed using MorphoGraphX ​​software according to the user manual (http: / / www.morphographx.org). In brief, for each shoot apical meristem, a surface mesh was extracted from the confocal image stack. Cells were then segmented and their size quantified. To assess cell growth and mitotic activity, confocal image stacks acquired at different time points from the same shoot apical meristem were segmented, and daughter cells were labeled with their parent cells. Cell growth heatmaps were created based on the size changes of the same cells at different time points. Cell division was analyzed using the "Proliferation Heatmap" tool.

[0154] 4. Atomic Force Microscopy (AFM) Measurement

[0155] AFM data collection was performed according to the previously described method (Peaucelle et al., 2011). Dissected apical meristems (SAMs) were fixed onto slides with room-temperature agar. Turbulence was eliminated by immersing the SAMs in 0.55 M mannitol. To measure the rigidity of the sample cell walls, the samples were indented in a 100 x 100 μm square area using an AFM cantilever equipped with a spherical needle tip. 64 x 64 measurements were performed within this area, for a total of 4096 force-indentation experiments. The apparent Young's modulus was calculated for each force-indentation experiment using the Hertz indentation model. Each pixel on the hardness map represents the apparent Young's modulus at one force-indentation point. For topographic reconstruction, the height of each point was determined by the contact point of the force-indentation curve; each contact point was derived from the point used to determine the apparent Young's modulus (E). A The same curve. The hardness data was projected onto the topographic map using Matlab. The cantilever used was: "Nano World" (NanoWorld AG, The TL-NCH-20 needle tip from Switzerland has a spring constant of 10 to 130 N / m (estimated at 1.5 N / m) and a spherical tip with a radius of 900 to 1100 nanometers.

[0156] 5. Co-immunoprecipitation (Co-IP)

[0157] To verify the interactions between proteins in plants, an immunoprecipitation experiment was performed. A 3,774 bp genomic fragment containing the CSLD5 coding sequence was amplified using primers D5g_F1(KpnI) / D5g_R1(BamHI) and cloned into the vector pUBQ10::eGFP(N)-CSLD5-NOS via homologous recombination, constructing pUBQ10::eGFP(N)-CSLD5-NOS. Genomic fragments of CESA1, CESA3, and CESA6 were amplified using primer pairs CESA1-flag-F(KpnI) / CESA1-flag-R(PstI), CESA3-flag-F(KpnI) / CESA3-flag-R(PstI), and CESA6-flag-F(KpnI) / CESA6-flag-R(PstI), respectively. The vector pCAMBIA1300-UBQ10-3xFlag(N)-NOS was digested with KpnI and PstI enzymes and inserted into the above PCR product through homologous recombination to construct pUBQ10::3xFlag(N)-CESA1-NOS, pUBQ10::3xFlag(N)-CESA3-NOS and pUBQ10::3xFlag(N)-CESA6-NOS.

[0158] All plasmids were transformed using Agrobacterium GV3101 and expressed in *N. benthamiana* leaves via co-infection. Infected leaves were ground into a fine powder in liquid nitrogen. An IP extraction buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% (v / v) glycerol, 1% (v / v) Triton X-100, and a mixture of protease inhibitors was added to the tissue powder, and the mixture was incubated at 4°C for 1 hour. Subsequently, the mixture was centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was collected and incubated with Anti-GFP Affinity Beads 4FF (Smart-Lifesciences) at 4°C for 2 hours. After washing the beads three times with IP extraction buffer, SDS loading buffer was added, and the immunoprecipitated proteins were detected using the appropriate antibodies.

[0159] 6. Co-location test of CESA and CSLD5

[0160] To achieve transient co-expression in *N. benthamiana*, pUBQ10::GFP-CESA1, pUBQ10::GFP-CESA3, and pUBQ10::GFP-CESA6 were co-transfected with pUBQ10::mCherry(N)-CSLD5 into *N. benthamiana* leaf cells via co-infection. Fluorescence signals were detected 48 hours post-transformation using a laser scanning confocal microscope (ZEISS LSM 880).

[0161] In co-localization studies of Arabidopsis seedlings, the constructs pCSLD5::mCherry-CSLD5, pUBQ10::eGFP-CESA1, pUBQ10::eGFP-CESA3, and pUBQ10::eGFP-CESA6 were transformed into Col-0. Dual reporter gene lines co-expressing mCherry-CSLD5 and GFP-CESAs were generated through hybridization. Fluorescence signals of GFP and mCherry were detected using a ZEISS LSM 880.

[0162] 7. Protein Expression and Purification

[0163] The DNA-binding domain coding sequences of MYB3R1 and MYB3R4 were amplified using primers R1-DBD-F / R and R4-DBD-F / R, respectively. All PCR products were inserted into the pTolo-EX1 vector via homologous recombination to obtain pTolo-EX1-MYB3R1-N and pTolo-EX1-MYB3R4-N. The constructs were transformed into E. coli (Rosetta, DE3) and cultured in LB medium at 37°C until OD600 = 0.5. Recombinant protein expression was induced by the addition of 0.5 mM isopropyl β-D-thiogalactoside (IPTG). Protein purification was performed after culturing the bacteria at 18°C ​​for another 20 hours. The recombinant protein was purified using Ni-NTA affinity beads (smart) according to the manufacturer's instructions.

[0164] 8. Yeast two-hybrid (Y2H) experiment

[0165] Yeast two-hybrid assays were performed using the DUAL hunter system (provided by Dualsystems Biotech) to detect protein-protein interactions. The coding sequences for CESA1, CESA3, and CESA6 were fused with the Nub fragment in the pXGY17 vector, while the complete coding sequence for CSLD5 was fused with the Cub fragment in the pXGY18 vector. These constructs were transformed into yeast strain NMY51 according to the manufacturer's instructions. The yeast transformants were grown on nutrient-limited media to examine interactions between different protein combinations.

[0166] 9. Electrophoretic Mobility Assay (EMSA)

[0167] DNA fragments obtained from the CSLD5 promoter region were labeled with Cy5 and used as probes for electrophoretic mobility assays. First, the DNA fragments were amplified using gene-specific primers, followed by a second round of PCR with Cy5 labeling at the 5' end using universal primers. The DNA-binding domains of MYB3R1 (amino acids 1-200) and MYB3R4 (amino acids 1-200) were purified and incubated with 7.5 nM Cy5-labeled DNA probes at room temperature for 30 minutes in a buffer containing 100 mM Tris-HCl (pH 7.6), 250 mM KCl, 25 mM MgCl2, 10 mM EDTA, and 10 mM DTT. In the competition assay, different amounts of unlabeled DNA with sequences identical to the fluorescent probe were mixed with the labeled probe. Subsequently, the protein-DNA mixtures were electrophoresed at 100 V for 1 hour at 4°C in a 4.5% native gel containing 0.5 × TBE. Gels were analyzed using a Typhoon FLA 9000 (FUJIFILM FLA 9000 plus DAGE).

[0168] 10. Assay for transcriptional activation activity

[0169] The transcriptional activities of MYB3R1 and MYB3R4 on the CSLD5 promoter were evaluated using a dual-luciferase reporter system. pCSLD5::LUC, containing a 2,995 bp CSLD5 promoter sequence, was obtained by digesting the SK-D5pro construct with KpnI and BamHI and ligating it into the pGreenII 0800-LUC vector. In these constructs, the REN gene was expressed under the 35S promoter and served as an internal control. UBQ10::GFP-MYB3R1, UBQ10::GFP-MYB3R4, and pCSLD5::LUC were transiently transfected into tobacco leaf cells using the Agrobacterium infiltration method, and the RNA silencing repressor P19 was co-expressed to improve gene expression efficiency. LUC and REN activities were measured using a Promega dual-luciferase reporter system. LUC activities were normalized relative to REN activities (LUC / REN), and their mean and standard error were calculated from six independent biological replicates.

[0170] 11. Chromatin Immunoprecipitation-qPCR (ChIP-qPCR)

[0171] For chromatin immunoprecipitation experiments, approximately 1000 apical meristems from pMYB3R1::GFP-MYB3R1 and pMYB3R4::GFP-MYB3R4 plants were collected for chromatin extraction. After grinding in liquid nitrogen, the plant material was cross-linked with 1% formaldehyde. ChIP was performed using an anti-GFP antibody (Ab290, Abcam) in a buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and a 1× protease inhibitor mixture, binding to protein A and protein G Dynabeads (Thermo Fisher Scientific, 510001D and 10003D) for immunoprecipitation. DNA was extracted using AMPure beads. qPCR was performed using primers D5-ChIP-R1-F / R and D5-ChIP-R2-F / R; primer sequences are listed in Table S2.

[0172] 12. mRNA in situ hybridization

[0173] For mRNA in situ hybridization, dissected shoot apical meristems were fixed overnight in FAA (3.7% formaldehyde, 5% acetic acid, 50% ethanol) at 4°C. Samples were then embedded in paraffin and cut into 8-micrometer thick sections. After dewaxing, rehydration, and further dehydration, the sections were hybridized with the CSLD5 RNA probe overnight at 55°C. After hybridization, the sections were washed in SSC and incubated with anti-digoxigenin-AP antibody (Roche) for 2 hours at room temperature. Hybridization signals were detected by NBT / BCIP (Roche) colorimetric reaction. The CSLD5 RNA probe was prepared by in vitro transcription using the DIG RNA Labeling Kit (Roche). Images were taken under a Zeiss AxioImager M2 microscope with a PlanApochromat 20× / 0.8NA objective and a Zeiss Axiocam MRc color camera.

[0174] For fluorescence in situ hybridization (FISH), anti-digoxigenin-POD (Roche) antibody was used instead of anti-digoxigenin-AP antibody, and the hybridization signal was detected using the TSA Plus Cy5 fluorescence system (Perkin Elmer). Before observing the hybridization signal, the nuclei of the sections were stained with 1 mg / ml DAPI. RNA FISH images were taken under a Zeiss LSM700 confocal microscope using a 20 × 0.8 NA dry objective lens, with laser excitation wavelengths of 405 nm (DAPI) and 633 nm (Cy5). Fluorescence intensity was quantified in Fiji.

[0175] 13. RNA sequencing analysis

[0176] For transcriptome analysis, shoot apical meristems from Col-0, csld5, and csld5;pATML1::GFP-CSLD5 plants were collected for RNA extraction and library construction. RNA sequencing was performed using the Illumina platform. Raw sequencing reads were pruned using Trimmomatic v0.39 to remove low-quality bases and adapter sequences. FastQC v0.12.1 was used to assess sequencing data quality. Reads were aligned to the TAIR10 transcriptome using Hisat2 v2.2.1. Gene expression levels were quantified using TPMCalculator v0.0.3 and featureCounts v2.0.6. Heatmaps were generated in R to visualize expression levels. GO enrichment and KEGG pathway analysis were performed using the ClusterProfiler package in R. Coding sequences (CDS) were obtained from TAIR and aligned to the nr protein database using DIAMOND BLASTX. An E-value threshold of 10^6 was used for each sequence. -3The first 20 BLAST hits were imported into Blast2GO Basic for GO mapping and annotation, with the default annotation configuration set to a GO weight of 5 and an annotation truncation value of 75. KEGG Orthology (KO) annotation was then performed using KofamKOALA by comparing sequence BLAST with KEGG GENES.

[0177] 14. Statistical Analysis

[0178] Quantitative analyses of shoot apical meristem (SAM) size, cell size, and plant weight were performed using GraphPadPrism 8 software (GraphPad Software, San Diego, CA). All values ​​are presented as mean ± standard deviation. Significance was assessed using Student's t-test or one-way ANOVA. Individual data points are presented as box plots, with error bars representing maximum and minimum values ​​(the center line represents the median; the upper and lower limits of the box are the upper and lower quartiles, respectively).

[0179] 15. Sequence

[0180] The amino acid sequence of the CSLD5 protein in Arabidopsis thaliana (SEQ ID NO:1)

[0181]

[0182] The amino acid sequence of the CSLD4 protein in rice (Oryza sativa) (SEQ ID NO:2)

[0183]

[0184] Example 1: Disturbance of cell wall homeostasis affects the development of shoot apical meristem.

[0185] Cell wall synthesis and remodeling are primarily mediated by glycosyltransferases (GTs). Although the Arabidopsis genome encodes a large number of GT genes (approximately 560; http: / / www.cazy.org), RNA sequencing and mRNA in situ hybridization experiments have revealed that only a subset of GT genes are expressed in the shoot apex meristem, and their mRNAs accumulate in specific regions and cell types (Yang et al., 2016). To investigate the function of cell wall synthesis in SAM development, the inventors selected 29 GT genes expressed in the meristem, which are involved in the synthesis of various cell wall components (…). Figure 1 (a) Among these genes, FUT1 showed high and uniform expression, representing type 1 as defined in the inventors' previous study (Yang et al., 2016); for type 2 (floral primordia enrichment), the inventors selected 6 genes from the GT29, GT31, GT34, and GT77 families; CSLD5 and GALS2 were the only two genes classified as type 3 (expressed only in dividing cells); for type 4 (widely expressed, but at higher levels in dividing cells), the inventors analyzed 19 genes belonging to six different GT families, including multiple GAUT and GATL members. Additionally, the inventors analyzed the expression pattern of GATL6, which was classified as type 5 (proto-cambium expression). For these 29 GT genes, the inventors isolated homozygous T-DNA insertion mutants with a Col-0 background, preferentially selecting those with insertion sites located in exons, and confirmed the T-DNA insertion using gene-specific identification primers.

[0186] SAM images of these mutants were collected using 3D laser scanning confocal microscopy and quantitative analysis was performed. Figure 1(b) The SAM size of most GT mutants is comparable to that of the wild type (Col-0), suggesting possible functional redundancy. However, the inventors observed slightly larger meristems in the xylan synthesis defective mutant cslc8 (Kim et al., 2020), as well as slightly larger meristems in the irx10-l, irx14-l, and f8h mutants with reduced glucuronide xylan content (Brown et al., 2009). In contrast, the meristems of csld5, xxxt2, gaut9, gaut10, gals2, and gals3 plants were significantly smaller than those of the wild type. The reduced meristem size in the xxxt2 single mutant is consistent with the dominant activity of XXT2 in xylan synthesis (Zabotina et al., 2012). GAUTs encode galacturonyltransferases, which mediate pectin synthesis by transferring galacturonic acid from UDP-galacturonic acid diphosphate to polygalacturonic acid. Glycosyl residue composition analysis revealed reduced GalA content in the gaut9 and gaut10 single mutants (Caffall et al., 2009), which may be one reason for the lower SAM content in these two mutants. The β-1,4-galactosyltransferase GALS transfers galactose from UDP-galactose to β-1,4-galactpentasaccharide, thereby participating in the synthesis of pectin rhamnose galacturonic acid I. The reduced meristematic tissue in the gals2 and gals3 mutants is consistent with the reduced Gal content in the single gals mutant (Liwanag et al., 2012).

[0187] In summary, the inventors' genetic screening identified cell wall components, particularly pectin and CSLD5 products, as key regulators of shoot apical meristem growth.

[0188] Example 2: CSLD5-mediated synthesis and expansion of new cell walls

[0189] Previous studies have shown that pectin controls cell wall mechanics and meristem morphogenesis (Peaucelle et al., 2011), but the functions of CSLD family proteins in stem cell growth remain unclear. To elucidate the cellular basis of csld5 meristem growth defects, the inventors reconstructed 3D images of wild-type (WT) and csld5 SAMs in Arabidopsis thaliana. Using MorphoGraphX ​​software, the cell size in the L1 layer was quantified (Barbier de Reuille et al., 2015), revealing that csld5 cells were significantly larger than wild-type cells. Figure 2(a) Cell swelling in csld5 SAM may suggest a faster growth rate. Therefore, the inventors introduced the plasma membrane marker pUBQ10::myr-YFP into WT and csld5 and performed time-series in vivo imaging of the apical meristem at 12-hour intervals. Consistent with previous findings, peripheral zone (PZ) cells in WT SAM grew faster than central zone (CZ) cells due to the wall elasticity characteristic of this region (Milani et al., 2011; Peaucelle et al., 2011; Kierzkowski et al., 2012). Conversely, this heterogeneous growth pattern was lost in csld5, with most epidermal cells, especially in the peripheral regions, expanding less than those in WT SAM. Figure 2 (b). Furthermore, the anisotropic growth of csld5 cells was also affected. Figure 2 c) indicates that CSLD5-mediated cell wall synthesis contributes to both cell growth and pattern formation.

[0190] Plant cell wall construction involves two phases: the formation of the cell plate during cell division, and the addition of new material to the existing cell wall during growth (Fecette et al., 2019). Compared to WT, the inventors found that the formation of new cell walls was significantly delayed in csld5. Within 24 hours, 20% of WT SAM cells showed newly formed cell walls, while this proportion was only 12% in csld5. Figure 2 (d). Previous reports have indicated that csld5 exhibits cell wall fragmentation in root cortex cells, a phenotype further exacerbated in csld2csld5 or csld3csld5 double mutants (Gu et al., 2016). Similar cell wall gaps have also appeared in leaf epidermal cells of the maize csld1 mutant (Hunter et al., 2012). However, the inventors found that this type of cell wall fragmentation is relatively rare in csld5 SAM cells. Figure 3 (a) Even in the severely growth-defective csld2csld5 double mutant, the cell walls of newly dividing cells remain intact. Figure 3 (a) This indicates that the response of shoot apical stem cells to damaged cell wall synthesis differs from that of root or leaf cells. Based on these results, the inventors hypothesize that CSLD5 promotes the growth of shoot apical meristem by facilitating the synthesis of new cell walls in dividing cells and the expansion of interphase cell walls.

[0191] Example 3: CSLD5 maintains the mechanical integrity of the cell wall

[0192] Next, the inventors evaluated the cell wall properties of csld5 in Arabidopsis thaliana. Using atomic force microscopy (AFM), they observed a significant decrease in the Young's modulus (EYoung) of the antislope wall of csld5 SAM dividing and interphase cells. Figure 3 b; Cohen's d = 1.96), indicating decreased cell wall rigidity in csld5. Consistent with the reduced cell wall rigidity, RNA sequencing results showed that 18.8% (117 / 622) of the genes downregulated in csld5 SAM overlapped with the stress core transcriptome. Figure 3 (c and d) (Van Moerkercke et al., 2019). Of these 117 CSLD5-dependent mechanical stress response genes, 22 belong to the 51 genes most susceptible to inducing mechanical stress responses (Lee et al., 2005). These genes include stress response marker genes TCH2, TCH3, and TCH4, as well as genes involved in calcium ion signaling, ethylene activity, and hypoxia response ( Figure 3 (d). In summary, the inventors' data demonstrate that CSLD5-mediated cell wall synthesis and transcriptome exhibit a certain degree of specificity and are partially correlated with mechanical stress response.

[0193] Example 4: CSLD5-mediated cell wall synthesis and feedback mechanisms in the cell cycle

[0194] In the inventors' mRNA in situ hybridization screening, CSLD5 and GALS2 were the only two GT genes exhibiting a patchy expression pattern regulated by the cell cycle (Yang et al., 2016). Through analysis of the CSLD5 promoter, the inventors identified five mitosis-specific activating elements (MSAs), which were recognized by the MYB3R transcription factor in chromatin immunoprecipitation sequencing (ChIP-seq) assays. Figure 4 (a and b) (Yang et al., 2016). The inventors further verified the binding of MYB3R1 and MYB3R4 to the CSLD5 promoter in Arabidopsis thaliana using ChIP real-time quantitative PCR (qPCR) and electrophoretic mobility assay (EMSA). Figure 4 Dual-luciferase reporter assays showed that the activity of the CSLD5 promoter was significantly enhanced in the presence of MYB3R1, MYB3R4, or both, indicating that these transcription factors directly activate the expression of the CSLD5 gene. Figure 4 This conclusion is supported by qRT-PCR and RNA FISH experiments, which showed that CSLD5 expression levels were significantly reduced in the myb3r1 myb3r4 double mutant. Figure 4 (g and h).

[0195] Compared to mRNA, the CSLD5 protein has a wider distribution. Figure 4 CSLD5 protein is translated from prophase and accumulates on the forming cell plate during telophase. Figure 4 This is similar to observations in root and leaf dividing cells (Gu et al., 2016). After new cell wall formation, the CSLD5 protein is recycled and persists within the cell for several hours. Figure 4 Non-cell plate-localized CSLD5 protein forms a loop around a trans-Golgi network (TGN) labeled with VHA1-RFP. Figure 4 The kinetic localization pattern of CSLD5 is similar to that of primary cell wall CESAs (Miart et al., 2013), highlighting the dual role of CSLD5 in cell plate formation and subsequent cell wall expansion.

[0196] Cell growth and division are closely coordinated (Sablowski and Gutierrez, 2022). Although CSLD5 expression is regulated by cell division, the inventors found that CSLD5 expression also affects cell cycle progression in Arabidopsis thaliana. Compared to wild-type, the expression level of HIS4 mRNA in CSLD5SAM was significantly reduced (…). Figure 5 ,a), while the expression of CYCB1;2 increased ( Figure 5 (b) HIS4 is a cell cycle marker gene that marks G1-S transition cells, while CYCB1;2 is expressed only in the early pre-transition phase. Figure 5 Therefore, mutations in CSLD5 lead to cell capture during the early stages of mitosis before cell plate formation, resulting in a reduction in the SAM size of CSLD5. Figure 5 ,d).

[0197] Example 5: CSLD5 interacts directly with CESA1, CESA3, and CESA6.

[0198] Within the cellulose synthase superfamily, CSLDs show the highest amino acid sequence similarity to CESAs (Richmond and Somerville, 2000). All CESAs and some CSLDs, including CSLD2, CSLD3, CSLD4, and CSLD5, possess a conserved N-terminal ring-shaped zinc finger domain, predicted to be involved in protein complex formation (Kumar et al., 2022). Consistent with this, the inventors found that CSLD5 interacts with CSLD3 in yeast. Figure 6 Furthermore, CSLD2 and CSLD3 are functionally interchangeable with CSLD5 in regulating meristem size and plant growth. Figure 6 (bg).

[0199] To explore the potential interaction between CSLD5 and primary cell wall CESAs, the inventors conducted a yeast two-hybrid experiment. Using a membrane yeast two-hybrid system based on cleavage ubiquitin, the inventors detected a strong interaction between CSLD5 and CESA3 or CESA6, and a weaker interaction between CSLD5 and CESA1. Figure 7 (a). To verify these interactions in plants, the inventors co-expressed GFP-CSLD5 with FLAG-labeled CESA1, CESA3, or CESA6 in tobacco (Nicotiana benthamiana) leaves and performed immunoprecipitation experiments. The inventors found that all three CESAs could be co-immunoprecipitated with GFP-CSLD5 using a GFP-specific antibody. Figure 3 The results (e) indicate that CSLD5 is physically associated with CESAs in both yeast and plant cells.

[0200] Next, the inventors observed the cellular dynamics of CSLD5 and CESA proteins. In a transient expression system in tobacco, the inventors observed co-localization of mCherry-CSLD5 with GFP-CESA1, GFP-CESA3, or GFP-CESA6. Figure 7 (b) The inventors further generated stable transgenic plants co-expressing mCherry-CSLD5 with GFP-CESA1, GFP-CESA3, or GFP-CESA6. Consistent with previous reports (Gu et al., 2016), CSLD5 and CESA3 co-modified the cell plate, and the co-localization of CSLD5 with CESA1 or CESA6 on the cell plate was also evident. Extensive overlap between CSLD5 and CESA proteins was also observed in the putative Golgi stack. Figure 7 In summary, these data indicate a direct interaction and co-localization between CSLD5 and primary cell wall CESAs.

[0201] To investigate the functional relationship between CSLD5 and CESAs, the inventors conducted a two-way genetic complementation experiment. CSLD5 promoter-driven GFP-tagged CESA1, CESA3, and CESA6 were expressed in the csld5 mutant. Although CESA1 had little effect on csld5 growth, csld5;pCSLD5::GFP-CESA6 plants exhibited severe growth retardation. Figure 8 The phenotypes of csld2csld5 double mutants (Yin et al., 2011) or higher-order CESA mutants (Persson et al., 2007) suggest that ectopic expression of CESA6 in dividing cells may disrupt the normal function of CSCs or the CESA-CSLD complex. Conversely, GFP-CESA3 was found to completely rescue the growth defects of csld5. Figure 8 This indicates that CESA3 is functionally equivalent to CSLD5 in controlling SAM growth and plant development.

[0202] Therefore, both molecular and genetic assays suggest that CSLD5 is involved in the synthesis of cellulose or cellulose-like cell wall material. Further supporting this, immunolabeling experiments using the cellulose-binding module CBM4 showed a significant reduction in the content of non-crystalline cellulose in csld5SAMs. Figure 3 In summary, although the activity of CSLD5 in xylan and pectin biosynthesis cannot be ruled out (Bernal et al., 2007; Zhu et al., 2010), the inventors' data, along with the discovery that CSLD5 expression is induced by isoxazol (Manfield et al., 2004) and the role of CSLD3 as a UDP-glucose-dependent β-1,4-glucose synthase (Yang et al., 2020), suggest that CSLD5 has a similar function to CESAs in the construction of cellulose-based cell walls.

[0203] Example 6: Restoring cell wall mechanics and shoot apical meristem growth by restricting CSLD5 to L1 cells.

[0204] To date, the inventors' data have clearly established CSLD5 as a regulator of new cell wall synthesis and expansion in the shoot apical meristem, which is crucial for maintaining the mechanical integrity of the cell wall. In the non-dividing stolons of *Utricularia filamentosa* and hypocotyl cells of *Arabidopsis thaliana*, mechanosynthesis has become a mechanism for coordinating interstitial growth (Kelly-Bellow et al., 2023). When GFP-CSLD5 protein driven by the RPS5A promoter was expressed throughout the SAM of *Arabidopsis thaliana* (Weijers et al., 2001), the inventors found that it only partially restored the small SAM and growth defects associated with CSLD5. Figure 9 This suggests that interlayer or intercellular growth coordination mediated by the cell wall may exist in proliferating meristems.

[0205] To verify this hypothesis, the inventors used the ATML1 promoter in Arabidopsis thaliana to restrict CSLD5 expression to the epidermis (Takada et al., 2013; Meyer et al., 2017). The pATML::GFP-CSLD5 construct was transformed into the csld5 mutant. RNA fluorescence in situ hybridization (FISH) was performed. Figure 10 (a and b) and confocal microscopy analysis ( Figure 11 As shown in (a) and (b), GFP-CSLD5 mRNA and its protein were specifically detected only in L1 cells of SAM. The inventors found that GFP-CSLD5 expressed in the L1 layer completely restored the levels of csld5 in SAM and plant size (…). Figure 10 By segmenting csld5;pATML::GFP-CSLD5SAM cells, the inventors found that the enlarged L1 cells in csld5 cells recovered to wild-type levels. Figure 11 Furthermore, quantification of L2 layer cell size also showed complete complementarity (c). Figure 11 Besides cell size, the number of cells in the L1 and L2 layers of csld5;pATML::GFP-CSLD5 SAM plants was significantly increased, even exceeding that of wild-type SAM ( ,d). Figure 11 (e and f). Therefore, the expression of CSLD5 in epidermal cells completely restored the cell growth and proliferation defects of the csld5 mutant.

[0206] RNA sequencing analysis showed that transcriptomic changes in csld5 SAM were also restored in csld5;pATML::GFP-CSLD5 plants. Figure 12Specifically, 79.5% (93 / 117) of CSLD5 maintenance and mechanosensitive genes, including mechanosensitive genes upregulated in the nuclear-harder gip1-gip2 mutants (Goswami et al., 2020), were reversed back to wild-type levels by CSLD5 expression in the L1 layer. Figure 11 g; Figure 12 (b) Consistent with these molecular and cellular complementarities, atomic force microscopy measurements showed that csld5;pATML::GFP-CSLD5 SAM was harder than csld5 alone. Figure 12 The result (c)(Cohen's d=0.839476) indicates that the mechanical properties were also restored. In summary, these results suggest that CSLD5-mediated cell wall synthesis regulates the activity of inner layer cells in a non-cell-autonomous manner, promoting SAM growth.

[0207] Example 7: Epidermal-specific expression of OsCSLD4 promotes the growth of rice inflorescence meristems and seed yield.

[0208] In quantitative analysis, the inventors frequently found that GFP-CSLD5 expression in L1 cells not only restored the growth defect of csld5, but also that csld5;pATML::GFP-CSLD5 plants were even larger than wild-type Col-0 ( Figure 10 (e and f). This observation suggests that remodeling the epidermal cell wall may be a potential strategy to enhance shoot apical meristem activity and overall plant growth. To test this hypothesis, the inventors used rice (Oryza sativa), a grass, as their research subject. The CSLD5 homolog OsCSLD4 in rice is known to be expressed in dividing cells and is crucial for plant growth (Li et al., 2009; Yoshikawa et al., 2013). The inventors expressed GFP-OsCSLD4 in the wild-type background of Zhonghua 11 (ZH11) under the control of the epidermal-specific promoter ROC1 (Ito et al., 2002). RNA FISH ( Figure 13 a) and confocal imaging ( Figure 14 a) Confirmed that both the mRNA and protein of GFP-OsCSLD4 are confined to epidermal cells.

[0209] In rice, the shoot apical meristem of pROC1::GFP-OsCSLD4 plants was larger than that of wild-type plants and showed higher levels of HIS4 expression. Figure 13 (b) indicates stronger cell division activity. At maturity, these transgenic plants are similar in height to the wild type. Figure 13 (c and d), but the increased number of tillers suggests enhanced activity of axillary meristem ( Figure 14(b and d). Although the spike length of pROC1::GFP-OsCSLD4 is comparable to that of the wild-type plant ( Figure 13 (e), but the number of primary and secondary branches increases ( Figure 14 Therefore, the spikes of pROC1::GFP-OsCSLD4 produced more seeds than the control group ZH11. Figure 14 f). Given that the seed weight of pROC1::GFP-OsCSLD4 plants is difficult to distinguish from that of ZH11 ( Figure 13 The increased number of seeds suggests that regulating the expression of OsCSLD4 in the epidermis has the potential to improve rice grain yield.

[0210] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. The application of an agent that promotes the specific expression of CSLD5 or CSLD4 protein or gene in plant epidermis in promoting cell wall synthesis, improving the tissue mechanical properties of shoot apical meristem, and improving plant traits.

2. The application as described in claim 1, characterized in that, The cell wall synthesis includes: the synthesis of new cell walls, the expansion of interphase cell walls, the anisotropy of cell growth, the construction of cell plates during cell division, and the addition of new materials to existing cell walls during growth.

3. The application as described in claim 1, characterized in that, The improved tissue mechanical properties of the shoot apical meristem include: enhanced cell wall stiffness and rigidity, and increased expression levels of mechanical stress-responsive genes; Preferably, the enhancement of cell wall stiffness and rigidity includes: an increase in the Young's modulus of the anticlinal wall of dividing cells and interphase cells; Preferably, the mechanical stress response genes include: TCH2, TCH3, TCH4, CML23, CML38, CPK28, CPK32, WRKY53, WRKY40, EFR11, PSK3, NHL3, AT1G76600, or HSPRO2.

4. The application as described in claim 1, characterized in that, The improvement of plant traits includes: promoting the growth of plant shoot tip meristem, increasing the size of plant shoot tip meristem, increasing plant weight, increasing plant leaf area, promoting the growth and development of shoot tip meristem cells, increasing the number of cells in L1 and L2 layers, increasing the number of plant divisions, promoting the increase of plant branches, increasing the number of tillers, increasing the number of primary branches and secondary branches per spike, increasing the number of grains per spike, and promoting the increase of plant yield.

5. The application as described in claim 1, characterized in that, The plant epidermal-specific expression includes: plant L1 layer cell-specific expression, preferably in the L1 layer cells of the plant shoot apical meristem; Preferably, the method for plant epidermal-specific expression includes: using a plant epidermal-specific promoter to guide the specific expression of CSLD5 or CSLD4 protein or gene in the plant epidermis; More preferably, recombinant DNA technology is used to insert a plant epidermal-specific promoter into an expression vector to control the expression of downstream CSLD5 or CSLD4 protein coding sequences or genes; More preferably, the plant epidermal-specific promoters include: ATML1 promoter, ROC1 promoter, GlymaML1 promoter, PtrML1 promoter, SIHDZIV7 promoter, TaROC2 promoter, and / or ZmHDZIV6 promoter.

6. The application as described in claim 1, characterized in that, The reagents include: plant epidermal-specific promoters, CSLD5 or CSLD4 protein coding sequences or genes, recombinant nucleic acid molecules, vectors, and host cells; Preferably, The plant epidermal-specific promoters include: ATML1 promoter, ROC1 promoter, GlymaML1 promoter, PtrML1 promoter, SIHDZIV7 promoter, TaROC2 promoter, and / or ZmHDZIV6 promoter; The recombinant nucleic acid molecule includes: a plant epidermal-specific promoter, and a CSLD5 or CSLD4 protein-coding sequence or gene; more preferably, DNA or RNA; The vector includes: an expression vector; more preferably, the expression vector includes: a plasmid, an adenovirus vector, a lentivirus vector, or an adeno-associated virus vector.

7. The application as described in claim 1, characterized in that, The plant is a plant containing shoot apical meristem; Preferably, the plants include dicotyledonous plants and monocotyledonous plants; more preferably, the plants include: grasses, cruciferous plants, and legumes; even more preferably, the plants include: Arabidopsis thaliana, rice, corn, wheat, barley, sorghum, rye, oats, sugarcane, soybean, alfalfa, rapeseed, sugar beet, tomato, cotton, sunflower, poplar, eucalyptus, and willow.

8. A method for promoting plant cell wall synthesis, improving the tissue mechanical properties of shoot apical meristems, and / or improving plant traits, characterized in that, The method includes: specifically expressing the CSLD5 protein or gene in the plant epidermis; Preferably, the plant epidermal-specific expression is as described in claim 5; Preferably, the method of promoting plant cell wall synthesis, improving the tissue mechanical properties of shoot apical meristem and / or improving plant traits is as described in any one of claims 2-4; Preferably, the plant is as described in claim 7.

9. A method for identifying plant cell wall synthesis capacity, shoot apical meristem tissue mechanical properties and / or traits, comprising: Analyze the expression level or activity of the CSLD5 or CSLD4 gene in plants, and / or the expression level or activity of the CSLD5 or CSLD4 protein; if the expression level or activity of the CSLD5 or CSLD4 gene, and / or the expression level or activity of the CSLD5 or CSLD4 protein are normal, then the plant's cell wall synthesis capacity, shoot apical meristem tissue mechanical properties and / or traits are normal. Conversely, abnormalities occur in the plant's cell wall synthesis capacity, the histomechanical properties and / or traits of the shoot apical meristem; Preferably, the method of promoting plant cell wall synthesis, improving the tissue mechanical properties of shoot apical meristem and / or improving plant traits is as described in any one of claims 2-4; Preferably, the plant is as described in claim 7.

10. A method for screening substances that regulate plant cell wall synthesis capacity, shoot apical meristem tissue mechanical properties, and / or traits, comprising: (1) Add the candidate material to the system expressing CSLD5 or CSLD4; (2) Detect the system and observe the expression or activity of CSLD5 or CSLD4 therein. If the expression or activity is increased, it indicates that the candidate substance can be used to promote plant cell wall synthesis, shoot apical meristem tissue mechanical properties and / or improve plant traits; if the expression or activity is decreased, it indicates that the candidate substance can be used to inhibit plant cell wall synthesis, shoot apical meristem tissue mechanical properties and / or plant traits. Preferably, the method of promoting plant cell wall synthesis, improving the tissue mechanical properties of shoot apical meristem and / or improving plant traits is as described in any one of claims 2-4; Preferably, the plant is as described in claim 7.

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