Plants possess unique primary cell walls made of complex polysaccharides that play critical roles in determining intrinsic cell and organ size. How genes responsible for synthesizing and modifying the polysaccharides in the cell wall are regulated by microRNAs (miRNAs) to control plant size remains largely unexplored. Here we identified 23 putative cell wall-related miRNAs, termed as CW-miRNAs, in Arabidopsis thaliana and characterized miR775 as an example. We showed that miR775 post-transcriptionally silences GALT9, which encodes an endomembrane-located galactosyltransferase belonging to the glycosyltransferase 31 family. Over-expression of miR775 and deletion of GALT9 led to significantly enlarged leaf-related organs, primarily due to increased cell size. Monosaccharide quantification, confocal Raman imaging, and immunolabeling combined with atomic force microscopy revealed that the MIR775A-GALT9 circuit modulates pectin levels and the elastic modulus of the cell wall. We also showed that MIR775A is directly repressed by the transcription factor ELONGATED HYPOCOTYL5 (HY5). Genetic analysis confirmed that HY5 is a negative regulator of leaf size that acts through the HY5-MIR775A-GALT9 repression cascade to control pectin levels. These findings demonstrate that miR775-regulated cell wall remodeling is an integral determinant of intrinsic leaf size in A. thaliana. Studying other CW-miRNAs would provide more insights into cell wall biology.

The acquisition and utilization of cell walls have fundamentally shaped the plant lifestyle. While the walls provide mechanical strength and enable plants to grow and occupy a three-dimensional space, successful sessile life also requires the walls to undergo dynamic modifications to accommodate size and shape changes accurately. Plant cell walls exhibit substantial mechanical heterogeneity due to the diverse polysaccharide composition and different development stages. Here, we review recent research advances, both methodological and experimental, that shed new light on the architecture of cell walls, with a focus on the mechanical heterogeneity of plant cell walls. Facilitated by advanced techniques and tools, especially atomic force microscopy (AFM), research efforts over the last decade have contributed to impressive progress in our understanding of how mechanical properties are associated with cell growth. In particular, the pivotal importance of pectin, the most complex wall polysaccharide, in wall mechanics is rapidly emerging. Pectin is regarded as an important determinant for establishing anisotropic growth patterns of elongating cells. Altogether, the diversity of plant cell walls can lead to heterogeneity in the mechanical properties, which will help to reveal how mechanical factors regulate plant cell growth and organ morphogenesis.

Perennial trees must maintain stem growth throughout their entire lifespan to progressively increase in size as they age. The overarching question of the molecular mechanisms that govern stem perennial growth in trees remains largely unanswered. Here we deciphered the genetic architecture that underlies perennial growth trajectories using genome-wide association studies (GWAS) for measures of growth traits across years in a natural population of Populus tomentosa. By analyzing the stem growth trajectory, we identified PtoP4H9, encoding prolyl 4-hydroxylase 9, which is responsible for the natural variation in the growth rate of diameter at breast height (DBH) across years. Quantifying the dynamic genetic contribution of PtoP4H9 loci to stem growth showed that PtoP4H9 played a pivotal role in stem growth regulation. Spatiotemporal expression analysis showed that PtoP4H9 was highly expressed in cambium tissues of poplars of various ages. Overexpression and knockdown of PtoP4H9 revealed that it altered cell expansion to regulate cell wall modification and mechanical characteristics, thereby promoting stem growth in Populus. We showed that natural variation in PtoP4H9 occurred in a BASIC PENTACYSTEINE transcription factor PtoBPC1-binding promoter element controlling PtoP4H9 expression. The geographic distribution of PtoP4H9 allelic variation was consistent with the modes of selection among populations. Altogether, our study provides important genetic insights into dynamic stem growth in Populus, and we confirmed PtoP4H9 as a potential useful marker for breeding or genetic engineering of poplars.

In plants, endoreplication, the process where nuclear DNA replicates in the absence of mitosis, and remodeling of the primary cell walls are both coupled with cell expansion. However, the mechanisms by which these two processes coordinate to determine cell size remain largely elusive. Here, employing the tcpΔ7 septuple mutant disabling seven of the eight CIN-TCP transcription factors in Arabidopsis, we find that hindered endoreplication progression in tcpΔ7 whereby ploidy increases from 8 C to beyond is correlated with an increase in cell wall pectin. CIN-TCPs transcriptionally activate POLYGALACTURONASE LIKE 1 (PGL1), which encodes a polygalacturonase downregulating both abundance and molecular mass of pectin polymers. Genetic analysis of PGL1 in both the wild type and tcpΔ7 backgrounds confirm that pectin reduction promotes endocycle progression and cell enlargement. Collectively, these findings reveal a critical role of pectin in regulating endoreplication, providing insights in the understanding of cell growth and organ development in plants.

Plants possess unique primary cell walls made of complex polysaccharides that play critical roles in determining intrinsic cell and organ size. How genes responsible for synthesizing and modifying the polysaccharides are regulated by microRNAs (miRNAs) to control plant size remains largely unexplored. Here we identified 23 putative cell wall related miRNAs, termed CW-miRNAs, in Arabidopsis thaliana and characterized miR775 as an example. We showed that miR775 post-transcriptionally silences GALT9 , which encodes an endomembrane-located galactosyltransferase belonging to the glycosyltransferase 31 family. Over-expression of miR775 and deletion of GALT9 significantly enlarged leaf-related organs, primarily owing to increases in cell size. Monosaccharide quantification, confocal Raman imaging, and immunolabelling combined with atomic force microscopy (AFM) revealed that the MIR775A - GALT9 circuit modulates pectin level and elastic modulus of the cell wall. We further showed that MIR775A is directly repressed by the transcription factor ELONGATED HYPOCOTYL 5 (HY5). Genetic analysis confirmed that HY5 is a negative regulator of leaf size and acts through the HY5-MIR775A-GALT9 repression cascade to control pectin level. These results demonstrate that miR775-regulated cell wall remodeling is an integral determinant for intrinsic leaf size in A. thaliana and highlight the need to study other CW-miRNAs for more insights into cell wall biology.

Key traits, such as stomatal density, aperture, and pavement cell morphology, play a crucial role in plant physiology and ecology. However, manual quantification of these features is exceedingly labor-intensive and impedes research efficiency. While several automated analysis tools exist for examining stomata and pavement cells, they often fall short in providing both comprehensive functionality and user-friendliness. By integrating the latest detection and segmentation model, YOLOv11, with geometric algorithms, we developed a deep learning-powered tool, StomataQuant. It boasts an intuitive graphical user interface compatible with standard personal computers, enabling automated multi-task analysis and feature extraction for stomata detection, stomata and pores segmentation, and stomata and pavement cells segmentation. Its interactive editing interface offering manual correction significantly improved the efficiency of detection and data analysis. Systematic evaluations across diverse datasets demonstrate that StomataQuant exhibits exceptional concordance with manual measurements in stomata detection, stomata and pores segmentation, and stomata and pavement cells segmentation tasks. In practical applications, StomataQuant also yields conclusions consistent with manual measurements on both stomatal and pavement cell morphology. In the present study, we highlighted its powerful automated stomata and pavement cell detection with segmentation capabilities, and expect StomataQuant to significantly accelerate research advancements in plant physiology and ecology studies.