Leaves play a critical role in light capture during photosynthesis, gas exchange, and water transport in crops (Tsukaya, 2006). In particular, leaf shape is a key aspect related to the plant type and canopy structure, and it affects light interception by cotton crops (Du et al., 2009; Feng et al., 2016; Chen et al., 2021; Wang et al., 2021). L-D₁ is the main locus regulating the development of four major leaf shapes in upland cotton: super okra (L₂s), okra (L₂o), sub-okra (L₂u, L₂e), and normal (l₂ ) (Jiang et al., 2015). Map-based cloning showed that the L-D₁ locus was similar to the Late Meristem Identity 1 (LMI1) gene. Furthermore, virus-induced silencing of this gene in an okra leaf-shaped variety was sufficient to recover normal leaf formation (Chang et al., 2016; Andres et al., 2017). Sub-okra shape is the ancestral leaf shape of tetraploid cotton that gave rise to the okra allele, and the normal shape is derived from a mutant allele that came to predominate and define the leaf shape of cultivated cotton; however, the relationship between L₂u and L₂e is still unclear (Andres et al., 2017).

The leaf shape of most upland cotton varieties has been designated as normal. The introduction of other leaf shapes with variable indentation depths of the leaf blades has been associated with production advantages, such as accelerated flowering rate, early maturity, reduced boll rot and lint trash incidence, increased whitefly and pink bollworm resistance, and higher foliar chemical application efficacy (Jiang et al., 2015). However, different studies have obtained inconsistent results on the effects of leaf shape on insect resistance, yield, photosynthetic rate, and water-use efficiency (Wilson, 1990; Pettigrew et al., 1993; Jiang et al., 2015; Andres et al., 2016).

Over the past 60 years, cotton production in China has developed rapidly owing to improvements in cultivars and farming technology (Dai and Dong, 2014; Yu et al., 2016). A high-yielding cultivation system has been established in the Yellow River Valley, which is one of the major cotton-planting areas in China (Dong et al., 2006, Dong et al., 2009). In this system, full-season cotton planting requires considerable material and labor inputs, which has decreased the sustainability of this planting mode in Yellow River Valley (Dai et al., 2017). Based on short-season cotton, a new alternative has been established to reduce labor and costs (Dong et al., 2010; Lu et al., 2017; Qi et al., 2021). Short-season cotton also plays a very important role in double-cropping systems, such as garlic–cotton, rape–cotton, and wheat–cotton systems, which have been widely adopted to increase land productivity and profitability in regions with relatively abundant water resources and near-optimum temperature conditions (Qi et al., 2018). Compared with full-season cotton, short-season cotton is characterized by a shorter growth and development period, compact growth form, and relatively concentrated flowering and boll-setting; thus, it can be sown late and still mature early (Yu et al., 2005). However, the shortened growth cycle places great demands on the plant type, maturity, and yield potential of newly developed cultivars (Qi et al., 2018).

Under high planting density, a normal leaf shape and compact growth pattern might negatively affect the canopy structure and compromise the potential improvements in fiber yield of short-season cotton. Our previous study showed that the sub-okra leaf shape improved the canopy structure and photosynthetic and physiological characteristics, thereby increasing the total biomass and yield of full-season cultivars (Jiang et al., 2023). Thus, we sought to verify whether sub-okra leaf shape might also confer its advantages to short-season cotton and increase the fiber yield.

Therefore, the main objective of this study was to determine the genetic origin of the sub-okra leaf shape and the effects of leaf shape under the control of the L-D₁ alleles on leaf anatomical characteristics, canopy structure, yield, and fiber quality in short-season cotton. Genomic differences of near-isogenic lines were analyzed to the identification of the introgressive segment using genome resequencing technology. Physiological traits such as the leaf area index (LAI), intercepted PAR rate (IPR), relative chlorophyll content (SPAD value), leaf net photosynthetic rate (Pn), and biomass of each organ were investigated to determine the biological process by which leaf shape affects short-season cotton yield. This study is expected to provide useful information and germplasm resources for the development of short-season cotton varieties with high photosynthetic efficiency.

Sub-okra leaf shape is regulated by L₂u in Gossypium hirsutum L. and L₂e in G. barbadense L. However, the relationship between L₂u and L₂e is still unclear. After analyzing the sequence of L₂ genes from the Gossypium genus, the results indicate that the sub-okra leaf is the ancestral leaf shape of tetraploid cotton that gave rise to the okra allele and that the normal leaf shape is a derived mutant allele that came to predominate and define the leaf shape of cultivated cotton (Andres et al., 2017). In the present study, the gene regulating sub-okra leaf shape was identified as L₂e in G. barbadense L. ( Figure 1 ). The 5′UTR and CDS sequence of this gene were consistent with that of the L₂u reported (Andres et al., 2017; Jiang et al., 2021). This result indicates that L₂e and L₂u may have a common genetic source; however, further evolutionary evidence is needed.

Compared to that of the normal leaf, the decrease in LA of the sub-okra leaf improved the canopy structure and increased the PAR of different canopy layers, indicating that the leaf could maintain higher photosynthesis ( Figures 3A–D ). LMA is a key feature that reflects many essential aspects of leaf economics and can be used to measure the leaf dry-mass investment per unit of light-intercepting leaf area deployed (Wright and Cannon, 2001). LMA is tightly related to thickness of a leaf blade or the density of tissues (Griffith et al., 2016). Species with a low thickness have a higher mesophyll conductance, which limits photosynthetic rate in many species and hence shapes variation in leaf morphology and anatomy (Muir et al., 2013). Compared to normal leaves, sub-okra leaves exhibiting a thinner leaf blade had a lower LMA, which might have contributed to the higher Pn ( Figures 3C, D, G , 8A–D ).

Leaf photosynthesis is determined by not only biochemical properties but also anatomical features. Lens-shaped epidermal cells can potentially focus light within the upper layers of a leaf, resulting in a high photo flux density, which is called focusing effect (Brodersen and Vogelmann, 2007). A more flattened epidermis could result in a reduced focusing effect (Xiao et al., 2016). Our results showed that the sub-okra leaf had a thinner ADET, which indicated that increased oblateness of the epidermis might relieve the focusing effect in sub-okra leaf ( Figures 5G, H ). The columnar palisade cells can minimize light scattering and enable light to penetrate a leaf, while the spherical spongy cells are more effective in scattering light and thus maximize light absorptance (Brodersen and Vogelmann, 2010; Xiao et al., 2016). In the present study, a decrease in thickness was also detected in both the palisade and spongy mesophyll, which allowed more light to be absorbed by leaves in the lower layers ( Figures 5K–N ).

Changes in canopy architecture that improve photosynthesis may enhance crop productivity (Feng et al., 2012a, Feng et al., 2012b; Huang et al., 2021; Xiao et al., 2021). Furthermore, any variations in leaf shape can significantly modify canopy architecture, which directly affects light interception and solar energy use (Feng et al., 2016). Okra and sub-okra leaf shapes might increase lobe depth and decrease lobe width, thereby changing the LAI and light penetration (Wells et al., 1986). Our previous results showed that okra and sub-okra leaf shapes effectively optimized the canopy structure of full-season cotton (Jiang et al., 2023). The results summarized herein showed that the LAI of lines with sub-okra leaves was higher than that of lines with okra leaves and smaller than that of lines with normal leaves in short-season cotton ( Figure 6 ). Concomitant with the decrease in LAI, the IPR was reduced in lines with sub-okra and okra leaf shapes, compared to normal leaves ( Figure 7 ). Although maximizing LAI is closely related to a higher yield, a suitable reduction in LAI might equally improve yield by increasing the light energy supply to the lower leaves and canopy radiation use efficiency (Song et al., 2013; Chang et al., 2019). With a modest reduction in LAI, sub-okra leaves improved total biomass and fruit biomass from different genetic backgrounds in full-season cotton (Jiang et al., 2023). Although the yield of lines with the sub-okra leaf shape also increased in short-season cotton, there were differences in biomass accumulation. As for short-season cotton, although the total biomass of line with normal leaf shape was greater than that of lines with okra and sub-okra leaf shape, fruit biomass in lines with sub-okra leaf shape was greater than that in lines with the other two leaf shapes in the normal climate year ( Figures 10A, C ; Supplementary Figures S6G , S7G ). In this study, compared to the normal weather condition of 2018, the increase in precipitation in August and the decrease in sunlight from June to August accounted for the formation of more biomass and ineffective bolls, which increased the biomass of fruit in line with normal leaf shape in 2020 ( Supplementary Figures S1 , S6G , S7G ; Figure 10B, D ). The results indicated that lines with sub-okra leaf shapes also produced a more effective yield than other lines with okra and normal leaf shapes in short-season cotton.

The Pn reflects the productivity potential of a genotype (Elmore, 1980). A strong relationship between Pn and yield has been demonstrated by elevated [CO₂] experiments (Bender et al., 1999; Mitchell et al., 1999; Ainsworth et al., 2004). Okra, sub-okra, and similar leaves (normal × okra) exhibited a higher photosynthetic rate than normal leaves (Wells et al., 1986). Semi-okra leaves (normal × super okra) also showed an increased photosynthetic rate at most growth stages (Zhu et al., 2005). In this study, Pn of sub-okra leaves also performed better than that of the other two leaf shapes in two genetic backgrounds of short-season cotton ( Figure 8 ). Therefore, we believe that the increase in Pn laid the foundation for yield improvement in lines with sub-okra leaves.

Pn is also determined by other biochemical properties of the leaves (Song et al., 2018). For example, chlorophyll is a key factor in the absorption of light energy. Chlorophyll content is closely related to productivity, which increases with the peak SPAD value (Feng et al., 2016). Our results showed that Pn and productivity of lines with sub-okra leaves increased with a slight decrease in SPAD value ( Figure 9 ). Some studies have reported that a decrease in chlorophyll content increased the efficiency of PSII and light incidence on the surface of leaves (Ort et al., 2011; Xiao et al., 2016; Song et al., 2017; Zhou et al., 2023) because the light-harvesting antenna rapidly and reversibly switched into a photoprotected quenched state, in which potentially harmful absorbed energy was dissipated as heat under conditions of excess sunlight (Alexander et al., 2007; Cutolo et al., 2023). The relatively lower chlorophyll content of sub-okra leaves might help alleviate the damage caused to the photosynthetic system by excess high-energy absorption and maintain higher photosynthetic efficiency in short-season cotton. However, this hypothesis must be further verified in future experiments.

Our results showed that sub-okra leaf shape also conferred the same serial advantages, such as decreasing the LMA, LAI, and chlorophyll content, and increasing the Pn in short-season cotton, in which the yield of fiber was improved, but the lint percentage and fiber quality were not affected ( Tables 1 , 2 ). The significant yield increase in L54 SUBOKRA indicated that the line with sub-okra leaves showed an even greater advantage on terms of yield in early maturing short-season cotton.

In this study, we mainly focused on the effect of sub-okra leaf shape on canopy and photosynthetic characteristics under a conventional plant density. Plant density is an important factor that regulates canopy light distribution and affects canopy photosynthetic capacity in field-grown cotton (Yao et al., 2016), and it can influence plant size and change leaf azimuthal distributions. The optimum LAI can normally be reached faster in dense plant populations than in sparse ones (Chapepa et al., 2020). Therefore, it is necessary to study the effect of sub-okra leaf shape on canopy structure and photosynthetic capacity under higher plant densities.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

HJ: Data curation, Formal analysis, Software, Validation, Visualization, Writing – original draft. XM: Conceptualization, Funding acquisition, Writing – review & editing. JS: Methodology, Investigation, Writing – original draft. MG: Methodology, Investigation, Writing – original draft. XZ: Methodology, Investigation, Writing – original draft. CZ: Methodology, Investigation, Writing – original draft. QC: Data curation, Formal analysis, Software, Validation, Visualization, Writing – original draft. YW: Project administration, Resources, Supervision, Writing – original draft. XW: Project administration, Resources, Supervision, Writing – original draft. JW: Project administration, Resources, Supervision, Writing – original draft. YC: Data curation, Formal analysis, Software, Validation, Visualization, Writing – original draft. DZ: Methodology, Investigation, Writing – original draft. FL: Methodology, Investigation, Writing – original draft. WZ: Methodology, Investigation, Writing – original draft. JZ: Conceptualization, Funding acquisition, Writing – review & editing.