Advancements in Rice Leaf Development Research

Rice leaf morphology is a pivotal component of the ideal plant architecture, significantly impacting rice yield. The process of leaf development unfolds through three distinct stages: the initiation of leaf primordia, the establishment and maintenance of polarity, and leaf expansion. Genes regulating leaf morphology encompass transcription factors, hormones, and miRNAs. An in-depth synthesis and categorization of genes associated with leaf development, particularly those successfully cloned, hold paramount importance in unraveling the complexity of rice leaf development. Furthermore, it provides valuable insights into the potential for molecular-level manipulation of rice leaf types. This comprehensive review consolidates the stages of rice leaf development, the genes involved, molecular regulatory pathways, and the influence of plant hormones. Its objective is to establish a foundational understanding of the creation of ideal rice leaf forms and their practical application in molecular breeding.


Introduction
Rice (Oryza sativa) is one of the most important cereal crops in the world and is also the staple food for nearly half of the world's population [1].However, the total area of arable land is limited; therefore, increasing grain yield per acre is an effective measure to alleviate population pressure and an important guarantee for national security [2].Rice yield can be increased by enlarging the leaf area and enhancing the photosynthetic capacity of the population.The uppermost three leaves of rice, especially the flag leaf, are considered the main source of assimilates for photosynthesis and biomass synthesis [3,4].Therefore, modifying the leaf shape of rice plays an important role in increasing yield [5].
Rice leaves are the primary organs for photosynthesis in plants, intricately linked with plant architecture and serving as a crucial indicator of morphological development [6].They can be categorized into three types based on leaf position and role in overall plant development: basal leaves, transitional leaves, and cauline leaves.Basal leaves, situated near the base of the tiller nodes, provide nourishment for tillering, root development, and the differentiation of basal internodes, contributing to the formation of robust stems and large panicles.Transitional leaves play a pivotal role in root growth, stem elongation, spikelet primordium differentiation and development, and grain formation.Cauline leaves, comprising the uppermost three leaves, are instrumental during grain filling and maturation [4].The size, thickness, drooping tendency, and leaf angle directly impact the leaf area index and canopy photosynthetic efficiency, thereby influencing rice yield [7].
Rice leaf development unfolds in three stages: the initiation of leaf primordia, the establishment and maintenance of polarity, and leaf expansion [8].Leaves originate from the shoot apical meristem (SAM), where the central zone maintains the activity of stem cells, while the peripheral zone generates leaf primordia [9].The development of leaves along the proximal-distal, adaxial-abaxial, and medial-lateral polarity axes determine the ultimate length, width, and curvature of mature rice leaves [10].This process is Plants 2024, 13, 904 2 of 14 intricately regulated by numerous functional genes (Table 1), transcription factors, and environmental factors, collectively shaping the effective photosynthetic surface area and photosynthetic efficiency [7,11].In light of numerous studies on leaf development, this review synthesizes the complexities of leaf developmental processes, elucidating their underlying mechanisms [7,12,13].Furthermore, we shed light on the intricate regulatory network governing rice leaf development, with a particular focus on the involvement of plant hormones at various developmental stages.Through this endeavor, we aspire to catalyze advancements in the development of ideal plant architecture and the pursuit of high-yield rice breeding.

Rice Leaf Development Process
The initiation of leaf development takes place in the peripheral region of the SAM [11,45] (Figure 1).When the apex of a leaf primordium extends beyond the SAM, the SAM's base triggers the formation of another leaf primordium.The time interval between the emergence of two consecutive leaf primordia is known as the Plastochron (P) [46].Initially, the peripheral zone of the SAM forms a small protuberance, growing toward the apex and the opposite side of the SAM, resulting in the formation of a crescent-shaped leaf Plants 2024, 13, 904 3 of 14 primordium, denoted as P1 [47].This primordium undergoes a transformation into a hoodlike structure, termed P2, driven by rapid cell division and elongation at the apex and margin regions [11,45] (Figure 1).Subsequently, the leaf primordium's two margins overlap, enclosing the SAM in an elongated conical configuration, marking the commencement of the P3 phase [12].It is during this phase that the boundary between the leaf and leaf sheath is delineated [11,12].Notably, epidermal cells engage in periclinal division, resulting in the formation of a distinctive protuberance at the leaf sheath's base [7].This phase also witnesses significant activities within various internal tissues.With the emergence of leaf auricles and ligules, the leaf primordium differentiates into young leaves and experiences significant elongation, signifying the onset of the P4 phase [48].It is noteworthy that during the P1 to P4 stages, the elongation of the leaf sheath is temporarily suppressed.It is only after the leaf blade's elongation culminates that the leaf sheath experiences rapid elongation, denoting the P5 stage [11] (Figures 1 and 2a-d).This stage is succeeded by a sequence of events involving cell multiplication and differentiation, leading to the continuous expansion and thickening and ultimately culminating in the maturation of the leaf at the P6 stage [12,49] (Figures 1 and 2a).
Plants 2024, 13, x FOR PEER REVIEW 3 commencement of the P3 phase [12].It is during this phase that the boundary between leaf and leaf sheath is delineated [11,12].Notably, epidermal cells engage in periclina vision, resulting in the formation of a distinctive protuberance at the leaf sheath's bas This phase also witnesses significant activities within various internal tissues.With emergence of leaf auricles and ligules, the leaf primordium differentiates into yo leaves and experiences significant elongation, signifying the onset of the P4 phase [4 is noteworthy that during the P1 to P4 stages, the elongation of the leaf sheath is tem rarily suppressed.It is only after the leaf blade's elongation culminates that the leaf sh experiences rapid elongation, denoting the P5 stage [11] (Figures 1 and 2a-d).This s is succeeded by a sequence of events involving cell multiplication and differentia leading to the continuous expansion and thickening and ultimately culminating in maturation of the leaf at the P6 stage [12,49] (Figures 1 and 2a).The development process of rice leaf.Leaf primordium cells are differentiated in the pe eral region of SAM, which rapidly divides into a crescent-shaped leaf primordium, denoted as P this process, the adaxial-abaxial polarity of the leaf primordium is established.The process of e lishing polarity along the medial-lateral axis occurs during the transition from the P1 to the P2 of leaf development.The P2 leaf primordium is hood shaped.At the highest of this hood, the vas bundles of the leaf blade start to differentiate.At the same time, a younger leaf primordium aris its opposite side.Subsequently, the older leaf primordium is transformed into an elongated co configuration by cell division and cell elongation, marking the commencement of the P3 phase during this phase that the boundary between the leaf and leaf sheath is delineated.The leaf elon rapidly at the P4 stage, and the leaf sheath elongates at the P5 stage.Then, the tip of the lea emerges from the sheath.Finally, bulliform cells, stomatal cells, vascular bundles, etc., are diffe ated and mature, and the whole leaf extends from the leaf sheath and flattens out.It marks the mat of the leaves, termed P6.P1-P6 are used to divide the different stages of leaf primordium.its opposite side.Subsequently, the older leaf primordium is transformed into an elongated conical configuration by cell division and cell elongation, marking the commencement of the P3 phase.It is during this phase that the boundary between the leaf and leaf sheath is delineated.The leaf elongates rapidly at the P4 stage, and the leaf sheath elongates at the P5 stage.Then, the tip of the leaf also emerges from the sheath.Finally, bulliform cells, stomatal cells, vascular bundles, etc., are differentiated and mature, and the whole leaf extends from the leaf sheath and flattens out.It marks the maturity of the leaves, termed P6.P1-P6 are used to divide the different stages of leaf primordium.

Initiation of Leaf Primordia
In the early phases of leaf development, cells located in the periphery of the SAM undergo a notable acceleration in their division, resulting in the formation of leaf primordia [5,45].The initiation of these leaf primordia hinges on two intricately interconnected pathways: the Polar Auxin Transport pathway (PAT) and the Asymmetric Leaves1/Rough Sheath2/Phantastica (ARP)/Knotted1-like homeobox (KNOX) antagonistic pathway [50,51].At the heart of this initiation process lies the need for a precise concentration of auxins to induce leaf primordia formation [45,52].The PAT pathway is chiefly regulated by the auxin efflux carrier, PIN-FORMED1 (PIN1) proteins.PIN1 strategically localizes within the outermost epidermal cells of the meristematic tissue and serves as a conduit for the transport of auxins to the meristem's apex [45].The localized accumulation of auxin, driven by the PAT, dictates the specific location where leaf primordia sprout.Simultaneously, these newly formed leaf primordia consume auxins in their immediate vicinity, resulting in a distinct spacing between adjacent leaf primordia [50,53].
Within the ARP/KNOX pathway, antagonistic interactions between ARP and KNOX transcription factor families orchestrate the initiation of leaf primordia [54].In the plant kingdom, KNOX transcription factors serve as key regulators of two critical aspects: the formation and sustenance of the SAM and the establishment of the proximal-distal axis within leaves [55].Conversely, ARPs are primarily associated with cell differentiation processes occurring within leaf primordia [56].These ARPs include ASYMMETRIC LEAVES1 (AS1), ROUGH SHEATH2 (RS2), and PHANTASTICA (PHAN).Within this intricate regulatory network, prominent KNOX family members, such as SHOOT MERISTEMLESS (STM) and BREVIPEDICELLUS (BP), exhibit downregulated expressions during the initiation of leaf primordia [57].This downregulation is a fundamental prerequisite for subsequent cell or tissue differentiation to occur within the emerging leaf primordia.In rice, the SAM expresses STM and BP in a highly specific manner.STM suppresses the expression of AS1 within the SAM [58].Conversely, leaf primordia specifically express AS1, which, in turn, effectively inhibits BP expression.This intricate interaction ensures the smooth differentiation of peripheral region cells in the SAM [58,59].It is important to note that high auxin concentrations may potentially repress the expressions of BP and STM within leaf primordia [60].Consequently, a delicate balance of elevated auxin levels and upregulated AS1 expression becomes a critical requirement for the initiation of leaf primordia.In summary, the ARP/KNOX antagonistic pathway offers a dynamic framework characterized by antagonistic interactions that not only facilitate the growth of leaf primordia but also maintain a lower degree of cell differentiation within the SAM's peripheral region.
The Oryza sativa homeobox (OSH) gene in rice, a member of the KNOX family Class I, plays a crucial role in the formation and maintenance of the SAM.The expression of KNOX is positively regulated by cytokinins and the KNOX proteins themselves [59].For instance, OSH1 serves as a key regulator in SAM formation and maintenance by facilitating the catabolism of brassinosteroids (BRs) [14].When OSH1 expression is reduced, as observed in the shoot organization (sho) mutant during the early vegetative stage, the SAM becomes shorter and wider [15].This alteration accelerates leaf initiation, resulting in a more randomized leaf sequence.Leaves in this context exhibit unique characteristics, appearing filamentous or shorter and narrower [15].Another critical factor in this intricate dance is phospholipase A1 (PLA1), a gene encoding the cytochrome P450 CYP78A11.PLA1 likely plays a role in catalyzing substances that control leaf development [17].It is expressed in leaf primordia and regulates the pace of leaf initiation and maturation [17].In pla1 mutants, the SAM undergoes enlargement due to increased cell division, leading to shortened intervals between leaf emergence.Consequently, plants with pla1 mutations exhibit a significantly higher total leaf count, albeit with individual leaves being smaller and plant height reduced [17].Therefore, OSH and PLA1 are pivotal in preserving SAM functionality and ensuring the controlled development of leaf primordia.
Within Arabidopsis thaliana, the ERECTA family (ERf), including ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERL2, have emerged as key players in the process of leaf initiation [61].These genes are expressed throughout the entire process of the SAM and leaf primordia formation, exerting control over leaf initiation, SAM morphology development, inflorescence differentiation, and other critical processes [61].Notably, when examining a triple mutant, er erl1 erl2, an intriguing phenomenon unfolds.The SAM in this mutant exhibits a distinctive appearance-broader and flatter compared with that in the wild type [62].This morphological alteration is accompanied by a noteworthy reduction in the expression of PIN1 within leaf primordia [62].Consequently, the rate of leaf initiation significantly slows down, leading to a decrease in the total leaf count [62].In rice, the ERf comprises ERECTA1 (OsER1), OsER2, and OsERL.While the knockout of OsER1 and OsER2 affects inflorescence development in rice [63,64], it is important to highlight that, as of the present, there is a noticeable gap in research concerning the involvement of ERf genes in regulating rice leaf development.

Establishment and Maintenance of Polarity
Once leaf characteristics are established, leaf primordial cells transition from simple cell division to a process of cell differentiation [7].During this transformation, the leaf gradually develops three distinctive polarity axes: the proximal-distal, adaxial-abaxial, and medial-lateral axes [50,65,66] (Figure 2a).In rice, leaves are band shaped and the leaf sheath and leaf blade are bounded by the leaf collar, auricle, and ligule, which are located at the proximal axis and distal axis [67] (Figure 2a,d).The xylem, found nearer to the upper epidermis, defines the adaxial end, while the phloem, closer to the lower epidermis, characterizes the abaxial end.Between these two vascular bundles, bulliform cells are distributed [68] (Figure 2e).Significantly, the accurate establishment of polarity along the adaxial-abaxial axis serves as a prerequisite for the proper development of polarity along the proximal-distal and medial-lateral axes [51].Mutations affecting polarity formation along the adaxial-abaxial axis can lead to abnormal leaf shapes, such as needle-like or rod-like leaves [10].Therefore, the accurate development of three polarities is not only instrumental in shaping the leaf morphology but also serves as the foundational basis for normal leaf development.

Establishment of the Adaxial-Abaxial Polarity
The adaxial-abaxial polarity defines the orientation from the upper epidermis to the lower epidermis within a leaf (Figure 2a).Correctly establishing polarity along this axis is crucial for enabling normal leaf development, resulting in a flat and expansive leaf structure while also influencing leaf thickness [50].This polarity establishment coincides with the growth of the P1 leaf primordium toward the apex and the opposite side of the SAM [12] (Figure 1).In the context of leaf development, three genetic pathways are known to contribute to the establishment of polarity along the adaxial-abaxial axis: the homeodomain-leucine zipper (HD-ZIP) pathway, the AS1-AS2 pathway, and the small RNA pathway [50].HD-ZIP III transcription factors play a pivotal role not only in the development of the SAM but also in shaping the adaxial face of lateral organs [10].Signals originating from the SAM enhance the development of the adaxial face by activating HD-ZIP III, while the KANADI (KAN) and YABBY transcription factor families promote the development on the abaxial side [58].Interestingly, HD-ZIP III and KAN function in an antagonistic manner to regulate adaxial-abaxial polarity in leaves [58].In rice, members of the HD-ZIP III gene family, OSHB1, OSHB3, and OSHB5, participate in the initiation of leaf primordia and the development of polarity along the adaxial-abaxial axis, with their actions being dependent on auxins [10].Additionally, miRNA166 contributes to the establishment of adaxial-abaxial polarity by regulating the expression of OSHB genes [10].Another gene, OsHox32 (also referred to as OSHB4), influences leaf development by modulating the expression of YABBY genes [27].The AS1-AS2 pathway inhibits the expression of KAN, thereby suppressing the development on the abaxial side, while promoting the adaxial face's growth by enhancing the expression of HD-ZIP III [58,69].miRNA165/166 and trans-acting short-interfering RNA (ta-siRNA) exhibit opposite polarity distribution and jointly promote adaxial-abaxial polarity development through their antagonistic interplay [70].miRNA165/166 is primarily expressed at the abaxial face, contributing to abaxial face development by inhibiting the expression of HD-ZIP III genes, while ta-siRNA degrades auxin response factors (ARFs) on the abaxial side, thereby promoting abaxial face development [70,71].ARGONAUTE 1b (OsAGO1b) suppresses the expressions of miRNA and ta-siRNA, resulting in a significant upregulation of OSHB and OsARFs, which are essential factors for controlling adaxial-abaxial axis polarity development [28].These findings underscore the intricate interplay of these three regulatory pathways in shaping the adaxial-abaxial polarity during leaf development.

Establishment of the Medial-Lateral Polarity
The medial-lateral polarity, which extends from a leaf's midrib toward its sides, plays a vital role in determining leaf width [51] (Figure 2a).While research on the establishment of polarity along this axis remains relatively limited, a more comprehensive understanding has been gained regarding the development of leaf width following polarity establishment (Table 1).The process of establishing polarity along the medial-lateral axis occurs during the transition from the P1 to P2 stage of leaf development [12].At these stages, the leaf primordium encircles the SAM as it grows along the medial-lateral axis, ultimately giving rise to a mature leaf with distinct central and lateral regions (Figure 1) [12].Within the central region, the midrib serves as a sturdy structural element, providing support for the leaf and enabling efficient photosynthesis.This crucial midrib formation is orchestrated by genes belonging to the YABBY gene family, notably the DROOPING LEAF (DL) gene [22].DL induces cell proliferation in the central leaf region, guiding midrib development.The midrib formation defects occur, causing the leaf drooping in the DL knockout plant [22].The WUSCHEL-related homeobox (WOX) gene family, unique to plants, also contributes significantly to leaf development.For instance, OsWOX3 participates in gibberellin (GA) synthesis and signaling pathway-associated negative feedback regulation, impacting both leaf lateral growth and the formation of vascular bundles [72].Additionally, genes like NARROW LEAF2 (NAL2) and NAL3, which are paralogs and encode transcriptional activators of OsWOX3A, play a crucial role in the development of leaf margin structures [23].
The suppression of these genes results in the absence of leaf margin structures and narrower leaves [23].Within the OsWOX3B branch, the LATERAL SYMMETRY1 (LSY1) gene shares a similar role in regulating leaf development along the medial-lateral axis [24].lsy1 mutants result in an incorrect regulation of this axis, leading to the loss of either or both lateral parts of leaves and, consequently, the emergence of asymmetric leaf phenotypes [24].OsWOX4 plays a pivotal role in regulating the maintenance of stem cell activity in the shoot meristem [73].The downregulation of OsWOX4 leads to reduced endogenous cytokinin synthesis, cessation of vascular bundle differentiation, midrib formation defects, and ultimately, narrower leaves [25].In summary, the development of leaf margins and the midrib in rice is intricately regulated by OsWOX3, OsWOX4, and DL genes, collectively influencing leaf width and structure along the medial-lateral polarity.

Establishment of the Proximal-Distal Polarity
The proximal-distal polarity, which determines the length of a leaf, refers to the direction from the leaf base to its tip [51,74] (Figure 2a).This polarity comprises key components, including the leaf sheath at the proximal end and the leaf blade at the distal end [65].The leaf ligule, auricle, and collar collectively form the boundary between the leaf blade and the sheath [65] (Figure 2d).During the P3 stage, these boundary structures begin to take shape [12].Notably, the epidermis near the proximal end undergoes radial cell division, facilitating the separation of the leaf blade from the sheath [12].One essential gene in this process is Oryza sativa LIGULELESS1 (OsLG1), which encodes a protein containing the SQUAMOSA promoter-binding protein (SBP) domain.OsLG1 is primarily expressed in the collar region of young leaves [26].lg1 mutants result in the absence of collar, auricle, and ligule structures, causing the leaf blade to remain directly connected to the sheath [26].This leads to a reduced leaf angle, impacting the leaf's photosynthetic efficiency.
YABBY and WOX play significant roles in plant lateral organ formation and SAM function.YAB3 RNA interference (RNAi) plants result in curled and knotted leaves, indicating their potential involvement in rice leaf cell differentiation [72].The overexpression of OsWOX3 can suppress YAB3 expression, yielding defective leaf phenotypes similar to YAB3 RNAi plants [72].In rice, Class I KNOX members, including OSH1, OSH6, OSH15, OSH43, and OSH71, negatively regulate leaf development [59].Their expressions are inhibited when leaf primordia initiate at the SAM's edge.Ectopic expressions of these genes disrupt leaf development, causing abnormalities in the proximal-distal polarity development [59].For instance, osh1 mutants exhibit extended sheath regions or displacement of ligules and auricles and blurred SAM boundaries [14].Overexpressing OSH genes can lead to ligule and auricle loss or even leaf blade absence, resembling phenotypes of YAB3-RNAi and OsWOX3 overexpression plants [75].Studies have revealed that BOP1/2 genes in Arabidopsis thaliana express at the leaf proximal end and near the adaxial face, promoting the establishment of leaf proximal-distal polarity by inducing AS2 and suppressing KNOX expression [76].Oryza sativa BLADE-ON-PETIOLE (OsBOP) genes can activate the differentiation of proximal leaf sheaths and inhibit the differentiation of distal leaf blades.Triple mutants (osbop1/2/3) exhibit severe inhibition of sheath development, significantly larger leaf blades, and the absence of ligules and auricles [65].While it is established that OsBOP genes regulate proximal leaf sheath development in rice, the precise regulatory mechanisms remain unclear.What is certain is that the genes of OsLG1, YABBY, WOX, and KNOX are involved in the polarity development of the proximal-distal axis.

Leaf Expansion
Leaf development advances once the three polarity axes are established, with leaf primordia undergoing further transformation through cell expansion and division, eventually maturing into fully developed leaves [49].Critical to this process is the activity of the leaf's intercalary meristem, which enables leaf extension.Epidermal cells, such as bulliform cells and stomatal cells, begin differentiation from the leaf tip and extend toward the base [7,77].Simultaneously, vascular bundles mature, and sclerenchyma cells form out-side these bundles [12].These sclerenchyma cells and vascular bundles play pivotal roles in maintaining the flattened shape of rice leaves, and the development of sclerenchyma cells and bulliform cells can influence leaf curvature [68].Upon the completion of leaf elongation, the leaf sheath undergoes rapid elongation.As the leaf tip emerges from the sheath, nearly all internal and epidermal leaf structures are fully formed, with the exception of the proximal region [12].Throughout these intricate processes, numerous genes are instrumental in regulating leaf development (Table 1).For instance, genes like Abaxially Curled Leaf 1 (ACL1) and ACL2 influence leaf curvature by modulating the size of bulliform cells [78].OsHox32 overexpression impacts both the number and size of bulliform cells, resulting in leaf curling [27].Mutations in SHALLOT-LIKE1 (SLL1) and Semi-Rolled Leaf2 (SRL2) lead to a reduction in the number of sclerenchyma cells on the abaxial surface, causing leaf rolling [29,30].
Aberrant cell division or cell elongation can also significantly affect leaf development.Growth-regulating factors (GRFs) and GRF-interacting factors (GIFs) collectively regulate rice leaf growth through precise control of cell multiplication [20].OsMBK3, a member of the GIF family, positively regulates leaf size and internode elongation by influencing cell multiplication without altering cell size [21].STD1 and NAL8 impact leaf size by regulating cell division and, consequently, cell numbers [32,33].Additionally, OsARF19 and OsCCC1 influence leaf width by affecting cell size [34,35].
The plant's vascular system, comprising the xylem and phloem, substantially influences leaf development.The counts of both large and small vascular bundles, as well as the spacing between the small vascular bundles, directly influence the width of rice leaves.Vascular bundle development is under the control of genetic factors and endogenous hormone levels [7].Notably, OsWOX3 and OsWOX4 influence vascular bundle development by modulating GA and cytokinin levels, thus influencing leaf size [25,72].The NAL1 gene is primarily expressed in vascular tissues, regulating leaf size by influencing cell division, vascular bundle count, and PAT [31].Overexpressing NAL2 and NAL3 results in wider leaves with increased vascular bundles, in contrast to the phenotype observed in nal2/3 transgenic antisense plants [23].Lastly, the NAL9 gene encodes an ATP-dependent Clp protease proteolytic subunit and displays a narrow leaf phenotype throughout the entire growth period in its mutants [38].Overall, leaf development is a complex process that is regulated by many genes.Abnormalities in either gene may lead to leaf defects.

Auxins
Auxins, crucial plant hormones, play a pivotal role in orchestrating the initiation and growth of leaves within the SAM.They finely regulate these processes by affecting cell growth and division in the early stages of leaf development [51].Auxins at high concentrations can suppress KNOX gene expression, thereby triggering the formation of leaf primordia [51,79].The direction of auxin movement is facilitated by PIN proteins, which enable polar transport.When auxin inhibitors are applied, PIN protein activity is disrupted, halting leaf development [80].Conversely, the exogenous application of auxins to the SAM can reactivate and stimulate leaf development [80].During leaf primordium formation, auxins are transported from the adaxial face of the leaf primordium toward the SAM.This movement maintains a low auxin concentration at the adaxial face, ensuring the proper establishment and maintenance of leaf polarity [81].
Two key genes, NAL7 and NAL1, influence leaf width by modulating auxin biosynthesis and its PAT [31,39].NAL7 primarily governs leaf width by regulating auxin biosynthesis.Compared with wide types, nal7 mutants exhibit decreased auxin levels in the leaf base, resulting in narrower leaves [39].In contrast, nal1 mutants display reduced capacity for PAT, leading to fewer veins and narrower leaves [31].Studies have shown that OsCR4, a receptor-like kinase, contributes to auxin distribution and concentration gradients.Loss of OsCR4 function results in an aberrant distribution of auxin within leaves and has severe effects on vascular tissue formation and epidermal development.This leads to the formation of wrinkled, smaller leaves [40].Additionally, auxins may coordinate the development of the proximal-distal polarity and adaxial-abaxial polarity in leaves after the initiation of leaf primordia.High auxin concentrations affect distal leaf development and the formation of the vascular system [13].Disruptions in auxin transport or signaling pathways can lead to a shift in leaf development toward the abaxial end [13].However, recent research suggests that both adaxial and abaxial leaf surfaces perceive auxin similarly, indicating a more intricate regulatory network for auxins in leaf development [82].

Brassinosteroids (BRs)
Brassinosteroids play a vital role in SAM maintenance and collar formation by activating the expression of genes related to cell elongation and cell wall modification [14,51].OSH1 suppresses the BR content by inhibiting the signaling pathway and accelerating its inactivation to ensure the formation and maintenance of leaf primordia [14].In osh1 mutants and CYP734A RNAi plants, the BR catabolism pathway is inhibited.It results in premature cell differentiation, tissue hardening, boundary defects between leaf and sheath, and SAM and leaf primordium [14].
In rice, exogenous BR application influences the division and elongation of cells located at the junction between the leaf blade and leaf sheath at the adaxial face [83].This asymmetrical cell development between the collar's adaxial and abaxial sides results in the curvature of the collar and an increased leaf angle [83].This phenomenon underscores the intimate correlations of BR biosynthesis and signaling with leaf angle.The biosynthesis of BR primarily involves enzymes from the cytochrome P450 protein family.Genes responsible for encoding these enzymes have a direct impact on leaf angle regulation.Notable examples include OsDWARF11/D11, OsDWARF4, CYP90D2/D2, and OsCYP51G3 [84][85][86][87].Mutations in these genes hinder the elongation of parenchyma cells near the collar, leading to upright leaves [84][85][86][87].The BR signaling pathway involves receptor kinases, such as OsBRI1 and OsBZR1.Knockout of OsBRI1 and OsBZR1 disrupts the elongation of parenchyma cells near the lamina joint, causing leaves to be upright [88,89].OsBSK3 directly interacts with OsBRI1 and positively regulates BR signaling [90].Therefore, the biosynthesis and signaling pathways of BR exert a notable influence on the development of parenchyma cells located at the adaxial face of the leaf collar, consequently acting as positive regulators of the leaf angle in rice.

Cytokinins
The development of leaves, from initiation to full morphogenesis, is intricately tied to the delicate balance between auxins and cytokinins [91].Cytokinins, in particular, play a pivotal role in the establishment and maintenance of the SAM [92].LONELY GUY (LOG), responsible for activating cytokinins, is critical; its loss prematurely halts SAM development [16].On the contrary, increased cytokinin levels enhance SAM activity, profoundly influencing leaf development and the formation of floral meristems [64].Furthermore, the cytokinin oxidase/dehydrogenase (OsCKX) genes, including OsCKX1, OsCKX2, and OsCKX8 as suppressors, negatively regulate basal internode diameter, leaf dimensions, and grain size [93].However, OsCKX4 as a promotor positively influences these several characters [93].Notably, OsDOF11 directly regulates OsCKX4 expression levels, thus affecting cytokinin levels, and a loss of OsDOF11 function results in smaller leaves and reduced plant height [42].

Gibberellins (GAs)
GAs play a crucial role in the intricate process of rice leaf development.GAs primarily regulate cell differentiation and elongation [41].Any mutations in genes associated with the GA signaling transduction can significantly impact the size of plant organs [41].Throughout the course of leaf development, increased GA level correlates with both larger cell size and cell number in leaves, resulting in enlargement leaves [94].Conversely, decreased GA level or the plant's weaker sensitivity to GAs leads to smaller leaves and dwarfed Plants 2024, 13, 904 10 of 14 plants [94].In this context, the rice PLA1 and PLA2 genes come into play downstream in the GA signaling pathway, where they contribute to the regulation of leaf maturation [18].They also participate actively in the process of rice leaf elongation mediated by GAs, primarily by influencing cell proliferation [18].Furthermore, OsGA2ox6 plays a pivotal role by facilitating the breakdown of GAs.This action reduces the endogenous GA levels, resulting in the upregulation of OSH1 and TB1 expressions [41].The net effect is stunted plant growth, increased tillering number, and smaller leaves [41].Moreover, GIBBERELLIN INSENSITIVE DWARF1 (GID1) and GID2 exert control over plant height and leaf size by modulating the GA pathway [95,96].The intricate interplay between high cytokinin concentrations and low GA levels serves to maintain the undifferentiated state of stem cells in the SAM [97].This delicate balance effectively inhibits the initiation of leaves [97].KNOX genes play a dual role in this process.They positively regulate cytokinin biosynthesis and simultaneously negatively regulate GA synthesis by inhibiting GA20ox activity [79].This dual action ensures that the SAM's form and function are maintained as they should be.Therefore, gibberellin is involved in the regulation of leaf initiation and leaf size.
In conclusion, the interactions between plant hormones, such as auxins, cytokinins, and BRs, as well as their homeostasis and spatial signal transmission, can effectively control and maintain the dynamic balance of cell division and differentiation in the SAM and influence the leaf development process.

Prospects
The morphology of rice leaves encompasses various vital attributes, including length, width, thickness, curvature, and angle, collectively comprising the blade, collar, auricle, ligule, and sheath.As the primary site for photosynthesis, rice leaf morphology holds a profound sway over plant growth and development and, ultimately, yield [7]. Therefore, the optimization of leaf morphology becomes a pivotal undertaking in breeding highyield rice varieties with an ideal plant architecture.For an ideal rice plant, the top three leaves must ideally manifest characteristics of being lengthy, erect, slender, V-shaped, and robust in thickness.Length and erectness contribute to expanded leaf surface area, while slender leaves economize on space utilization.V-shaped leaves retain their tautness and are resistant to drooping, and thicker leaves manifest vigorous photosynthesis with reduced senescence, ultimately amplifying the overall exploitation of sunlight [6].These favorable leaf traits denote the plant's capacity to generate more carbohydrates, a cardinal prerequisite for achieving high yields.Hence, the crux of realizing ideal plant types lies in the synthesis of these favorable leaf traits, emphasizing the pressing need for deeper insights into rice leaf development processes and the intricate web of regulatory mechanisms.
The development of rice leaves stands as a multifaceted process, meticulously governed by numerous genes (Table 1), intricately intertwined with various hormones, and susceptible to the influence of environmental factors.Despite considerable strides in unraveling the regulatory mechanisms underpinning rice leaf development, much of this research remains compartmentalized.A comprehensive understanding of the intricate network of interactions among different genes involved in rice leaf development, alongside the delicate interplay between genes, hormones, and environmental cues, remains somewhat elusive.Furthermore, the knowledge landscape of rice leaf development is unevenly contoured, with a predominant focus on genes regulating leaf width, while leaf thickness has been relegated to relative obscurity.This inequity can be attributed, in part, to the inherent intricacy in quantifying leaf thickness characteristics.Yet, with the advent of advanced technologies, such as genome-wide association studies (GWAS) and single-cell sequencing, prospects loom large for a more lucid delineation of the regulatory network governing leaf development.Concurrently, the continued evolution and refinement of gene-editing technologies augur well for the sculpting and refinement of ideal leaf morphologies.These advancements are poised to significantly elevate the molecular design technology framework for breeding ideal rice plant types, ultimately ushering in substantial enhancements in rice yields.

Figure 1 .
Figure1.The development process of rice leaf.Leaf primordium cells are differentiated in the pe eral region of SAM, which rapidly divides into a crescent-shaped leaf primordium, denoted as P this process, the adaxial-abaxial polarity of the leaf primordium is established.The process of e lishing polarity along the medial-lateral axis occurs during the transition from the P1 to the P2 of leaf development.The P2 leaf primordium is hood shaped.At the highest of this hood, the vas bundles of the leaf blade start to differentiate.At the same time, a younger leaf primordium aris its opposite side.Subsequently, the older leaf primordium is transformed into an elongated co configuration by cell division and cell elongation, marking the commencement of the P3 phase during this phase that the boundary between the leaf and leaf sheath is delineated.The leaf elon rapidly at the P4 stage, and the leaf sheath elongates at the P5 stage.Then, the tip of the lea emerges from the sheath.Finally, bulliform cells, stomatal cells, vascular bundles, etc., are diffe ated and mature, and the whole leaf extends from the leaf sheath and flattens out.It marks the mat of the leaves, termed P6.P1-P6 are used to divide the different stages of leaf primordium.

Figure 1 .
Figure1.The development process of rice leaf.Leaf primordium cells are differentiated in the peripheral region of SAM, which rapidly divides into a crescent-shaped leaf primordium, denoted as P1.In this process, the adaxial-abaxial polarity of the leaf primordium is established.The process of establishing polarity along the medial-lateral axis occurs during the transition from the P1 to the P2 stage of leaf development.The P2 leaf primordium is hood shaped.At the highest of this hood, the vascular bundles of the leaf blade start to differentiate.At the same time, a younger leaf primordium arises on its opposite side.Subsequently, the older leaf primordium is transformed into an elongated conical configuration by cell division and cell elongation, marking the commencement of the P3 phase.It is during this phase that the boundary between the leaf and leaf sheath is delineated.The leaf elongates rapidly at the P4 stage, and the leaf sheath elongates at the P5 stage.Then, the tip of the leaf also emerges from the sheath.Finally, bulliform cells, stomatal cells, vascular bundles, etc., are differentiated and mature, and the whole leaf extends from the leaf sheath and flattens out.It marks the maturity of the leaves, termed P6.P1-P6 are used to divide the different stages of leaf primordium.

Table 1 .
List of genes that regulate rice leaf development.