GHCU, a Molecular Chaperone, Regulates Leaf Curling by Modulating the Distribution of KNGH1 in Cotton

Abstract Leaf shape is considered to be one of the most significant agronomic traits in crop breeding. However, the molecular basis underlying leaf morphogenesis in cotton is still largely unknown. In this study, through genetic mapping and molecular investigation using a natural cotton mutant cu with leaves curling upward, the causal gene GHCU is successfully identified as the key regulator of leaf flattening. Knockout of GHCU or its homolog in cotton and tobacco using CRISPR results in abnormal leaf shape. It is further discovered that GHCU facilitates the transport of the HD protein KNOTTED1‐like (KNGH1) from the adaxial to the abaxial domain. Loss of GHCU function restricts KNGH1 to the adaxial epidermal region, leading to lower auxin response levels in the adaxial boundary compared to the abaxial. This spatial asymmetry in auxin distribution produces the upward‐curled leaf phenotype of the cu mutant. By analysis of single‐cell RNA sequencing and spatiotemporal transcriptomic data, auxin biosynthesis genes are confirmed to be expressed asymmetrically in the adaxial‐abaxial epidermal cells. Overall, these findings suggest that GHCU plays a crucial role in the regulation of leaf flattening through facilitating cell‐to‐cell trafficking of KNGH1 and hence influencing the auxin response level.


Fig. S1
Fig. S1 Phenotypes of TM-1 and T582.(a) SEM images depicting leaf surfaces of parent plants.A single epidermal cell near the center is highlighted in green.(b) Statistical analysis of leaf area and cell size in TM-1 and T582.(c) Toluidine blue staining of paraffin sections using leaves from the parents.(d) SEM images showing the leaf primordium.The first true leaf of each parent is marked in green.AD refers to the adaxial side of the leaf, while AB refers to the abaxial side.Scale bars = 100 μm.P-values were determined using Student's t-test (**p < 0.01).

Fig. S2
Fig. S2 Relative expression level of the seven selected candidate genes were analyzed by qRT-PCR in (a) leaf margin and (b) leaf primordium.The data is presented as the mean (±SD) of three experimental replicates; Pvalues were determined using Student's t-test (*p < 0.05, **p < 0.01)

Fig. S4
Fig. S4 Spatial expression pattern of GHCU.(a) Analysis of GHCU expression level in different organs of the parents by qRT-PCR.The error bars in the graph represent SD of three biological replicates.(B)The expression level of AtCCT8 in Arabidopsis by qRT-PCR (c) GUS-stained whole plants of 6-week-old Arabidopsis.Magnification of the GUS-stained 5th true leaf (d) and inflorescence stem with flowers (e) of Arabidopsis.(f) Accumulation of GHCU transcripts, as visualized by RNA in situ hybridization of the SAM tissues of the parents.Primordium 1 (P1), Primordium 2 (P2), shoot apical meristem (SAM).Scale bars = 1 cm in (c), (d), and (e), and 100μm in (f).

Fig. S6
Fig. S6 Complementation of the Arabidopsis cct8 mutant through ectopic expression of GHCU.(a) The phenotypes of wild-type (WT) Arabidopsis, cct8 (SALK_082168c), and cct8 trans-formed with GHCU (C1 and C5).Scale bar = 0.5 cm.(b) The mRNA expression in WT, cct8 and the complementation, is analyzed using RT-PCR.Actin was used as the internal control.

Fig. S7
Fig. S7 Plant hormone content in tobacco and cotton using ESI-HPLC-MS/MS.(a) cytokinin (CK) and gibberellic acid3/4 (GA3/4) content in leaves from wild type SNN and three transgenic tobacco lines.(b) IAA content in leaves of three transgenic tobacco lines.(c) IAA content in leaves of three transgenic cotton lines.The p-values were determined using Student's t-test (**p < 0.01).

Fig. S8
Fig. S8 Ectopic expression of GHCU in Nicotiana benthamiana.(a) Photographs of tobacco plants overexpressing GHCUTM/T582 are shown.(b) GUS-stained intact leaves of transgenic tobacco.are presented, with pBI121-GUS is serving as the positive control.(c) mRNA expression of CK, GHCUTM-1, and GHCUT582, was analyzed using RT-PCR., with Actin was utilized as an internal control.(d) The IAA content in the leaves of the plants in (a)shown in (a) was measured.Scale bars = 5 cm.

Fig. S9
Fig. S9 Phylogenetic tree of three chrysanthemum KNOX proteins in Arabidopsis and KNGH1 in cotton.The yellow line represents the KNOX class I branch, the green line represents the KNOX class II branch, and the blue line represents the KNATM branch.

Fig. S10
Fig. S10 Ectopic expression of KNGH1 in Nicotiana benthamiana.(a) Photographs of tobacco plants overexpressing KNGH1.Abnormal leaves are marked with red arrows.(b) GUS-stained whole leaves of transgenic tobacco.pBI121-GUS was the positive control.(c) The IAA content in the leaves of the plants in (a).(c) mRNA expression of CK and KNGH1-GUS was analyzed using RT-PCR.Actin was used as an internal control.Scale bars = 5 cm.P-values were determined by Student's t-test (**p < 0.01).

Fig. S11
Fig. S11 GHCU is not enriched at the PD.Plasmolysis in the leaf epidermis does not show any noticeable enrichment of GHCU:GFP in the PD.The White arrow indicates the cell nucleus, while the asterisks indicate partial separation of the plasma membrane from the cell wall.Scale bars = 20 μm.

Fig. S12
Fig. S12 Division of seven anatomical shoot tip regions.

Fig. S13
Fig.S13The adaxial and abaxial epidermal areas are separately delineated on slices of two samples, leaf-ab: the SPOTs were chosen for abaxial side; leaf-ad: the SPOTs were chosen for adaxial side.

Fig. S15
Fig. S15 Violin plots showing the expression pattern of representative cell-specific marker genes used to assign cell types.