Leaf morphology is critical for the survival of plant species. After a leaf primordium is initiated at the flank of shoot apical meristem (SAM), the development along the medial‐lateral direction enlarges the leaf‐blades, leading to the increase of photosynthetic activities. Thus, the revelation of mechanisms that control development across a leaf is quite important for plant breeding. A variety of narrow leaf mutants have been identified in the grass family, which includes particularly important crops in the world. Here, the molecular mechanisms underlying the leaf development in the medial‐lateral direction are discussed as we introduce the three major groups of narrow leaf mutants in the grass family: (1) auxin‐related mutants, (2) cellulose synthase‐like D (CSLD)‐related mutants, and (3) polarity‐related mutants. The results obtained from these analyses could be directly applied to the breeding of major cereal crops such as maize, rice, and barley; therefore, they could contribute to the increase of food production.
- leaf morphogenesis
- gene expression
Leaves are the major photosynthetic organs in plants. The light‐capture efficiency significantly differs depending on the leaf shapes, angles, and arrangements in the canopy. Steeper leaf angle allows more light to penetrate to the lower leaves, leading to the increase of carbon gain through assimilation . To avoid self‐shading, leaf arrangement (phyllotaxis) is highly regulated by the plant hormone auxin [2, 3]. Since carbohydrates used in living activities are largely derived from the photosynthesis in plants, leaf morphology is critical for the survival of plant species.
A leaf primordium is initiated at the flank of shoot apical meristem (SAM), in which cells are maintained an indeterminate state by classIknotted1‐likehomeobox (KNOX) genes. The Arabidopsisthaliana genome includes four classIKNOX genes; shootmeristemless (STM), brevipedicellus(BP), KN1‐like in Arabidopsisthaliana2 (KNAT2), and KNAT6 . STM is expressed throughout the SAM and induces cytokinin biosynthesis via isopentenyltransferase7 (IPT7) activation and negatively regulates gibberellin biosynthesis via GA20‐oxidase1 (GA20ox1) repression . The resulting high cytokinin and low gibberellin ratio promotes meristem maintenance . Such STM expression is downregulated by plant hormone auxin [2, 3]. Auxin is unique in its polar transportation mediated by influx carriers represented by AUXIN1 (AUX1) and LIKE-AUX1 (LAX) proteins, and efflux carriers represented by PIN-FORMED (PIN) and ATP-binding cassette B (ABCB) proteins . Once transported to SAM, auxin flows to the peripheral young leaf primordia, creating an auxin maximum in the region where leaf primordia do not exist in the meristem. Such auxin localization downregulates STM expression, leading to the low cytokinin and high gibberellin ratio, which promote the switch from an indeterminate to a determinate state . The loss‐of‐function of PIN1 results in the malformed leaf development such as fused or cup‐shaped leaves, suggesting that localized auxin accumulation in the meristem determines the radial position of leaf initiation .
In SAMs, STM also downregulates the expression of the MYB transcription factor asymmetricleaves1 (AS1) and lateral organ boundaries domain (LBD) transcription factor AS2. When STM is repressed due to the auxin localization, AS1 and AS2, released from the negative regulation of STM, act together as a heterodimer to repress the expression of BP, KNAT2, and KNAT6 to prevent cell fate from returning to meristem [10–12]. The loss‐of‐function of AS1 resulted in the malformation of leaves due to the ectopic BP expression, which was enhanced with the additional loss‐of‐function of auxin resistant1 (AXR1) encoding a subunit of the related to ubiquitin1 (RUB1) activating enzyme that affects auxin responses . These results suggest that the expression of AS1 together with auxin localization plays a pivotal role in conferring leaf fate and promoting leaf development. Interestingly, slight KNOX expression remains in leaf primordia in species with compound leaves . In tomato, the classIKNOX genes tomatoknotted1 (TKN1) and TKN2 are expressed in young leaf primordia [15, 16]. The repression of TKN activity quickens the transition of the leaf primordia from the initiation to the secondary morphogenesis, suggesting that KNOX proteins are involved in the delay of leaf maturation and enable leaflet formation within leaf primordia .
The morphogenesis of sophisticated leaf organs with high reproducibility is achieved through the development in accordance with three axes; the proximal‐distal, adaxial‐abaxial, and medial‐lateral directions (Figure 1A–E) [8, 17]. The development along the medial‐lateral direction enlarges the leaf‐blades, leading to the increase of photosynthetic activities. Thus, the revelation of developmental mechanism along the medial‐lateral direction is quite important for plant breeding. So far, a variety of narrow leaf mutants have been identified in the grass family, which includes particularly important crops in the world. The results obtained from these analyses could be directly applied to the breeding of major crops such as maize, rice, and barley; therefore, they could contribute to the increase of food production. In fact, erect and narrow‐leafed rice mutants led to the higher photosynthetic CO2 uptake and improved yield in dense planting . Recently, it was revealed that the Quantitative Trait Locus (QTL) controlling flag leaf morphology and photosynthetic activity were allelic to the causal gene for narrow leaf mutant in rice, suggesting the availability of narrow leaf genes for breeding high‐yield varieties [19–23].
Here, the molecular mechanisms underlying the leaf development in medial‐lateral direction are discussed as we introduce the three major groups of narrow leaf mutants in grass family: (1) auxin‐related mutants, (2) cellulose synthase‐like D (CSLD)‐related mutants, and (3) polarity‐related mutants.
2. Auxin‐related narrow leaf mutants
Auxin is a fundamental plant hormone and regulates a variety of plant growth and development. All parts of the young plant such as cotyledons, expanding leaves, and root tissues can potentially produce auxin although the youngest leaves exhibit the highest biosynthetic capacity [24–26]. Auxin is unique in its polar transportation (polar auxin transport (PAT)), as we mentioned above, mediated by influx carriers and efflux carriers . The direction of auxin flow is the consequence of asymmetric localization of these carriers at plasma membrane [27, 28]. The resulting auxin localization within organs plays pivotal roles in phyllotactic patterning [29, 30], organogenesis [9, 31, 32], embryogenesis [33, 34], tropic response , and apical dominance . At the cellular level, auxin regulates cell division, cell elongation, and cell differentiation [7, 37].
The predominant form of auxin is indole‐3‐acetic acid (IAA). Genetic and biochemical analyses indicated that tryptophan (Trp) is the main precursor of IAA in plants, and four biosynthetic pathways for IAA from Trp have been assumed [38–40]. Among IAA biosynthetic enzymes revealed so far, the most important biosynthetic enzymes are the tryptophan aminotransferase of arabidopsis (TAA) family of aminotransferases and the YUCCA (YUC) family of flavin‐containing monooxygenases [41, 42]. TAA1 catalyzes the conversion of Trp to indole‐3‐pyruvic acid (IPA) in the initial step of the IPA pathway, and YUC catalyzes the conversion of IPA to IAA, downstream of TAA, in arabidopsis [40, 42–45]. The inactivation of a single TAA or YUC gene showed no obvious defects, indicating overlapping functions among TAA or YUC family members. On the other hand, the simultaneous inactivation of TAA1 and its close homologs, TAA‐related1 (TAR1) or TAR2 (Figure 2A), or inactivation of two or more YUC genes resulted in multiple growth defects together with a severe reduction in IAA level [43, 46]. Therefore, the IPA pathway, catalyzed by TAA and YUC, is considered to be the major auxin biosynthetic pathway in Arabidopsis .
The importance of the IPA pathway in IAA biosynthesis is also demonstrated in grass family. In maize, loss‐of‐function of vanishingtassel2 (VT2) and sparseinflorescence1 (SPI1), co‐ortholog of TAA1 and YUC in maize, respectively (Figure 2), caused severe barren inflorescences and semidwarf vegetative phenotypes with fewer leaves together with the reduction in IAA content [47, 48]. Similar reduction in IAA levels was shown in the loss‐of‐function of fishbone (FIB) and narrowleaf7 (NAL7), co‐ortholog of TAA1 and YUC in rice, respectively (Figure 2) [49, 50]. Thus, the IPA pathway seems to be the major IAA biosynthetic pathway in plants.
The reduction in IAA levels gives rise to pleiotropic organ malformation together with severe narrow leaf phenotype in rice. Tryptophandeficientdwarf1 (TDD1) encodes a protein homologous to the anthranilate synthase β‐subunit, which catalyzes the initial step of the Trp biosynthesis pathway . TDD1mutant is embryonic lethal because of a failure to develop most organs during embryogenesis. Regenerated TDD1 plants exhibit pleiotropic malformations including dwarfing, narrow leaves, short roots, and abnormal flowers, together with a reduction in Trp and IAA content. Trp feeding and moderate expression of OsYUC1 rescued the mutant phenotypes, indicating that abnormal phenotypes of TDD1 were caused mainly by Trp and IAA deficiency . The loss‐of‐function of constitutivelywilted1 (COW1), which encodes OsYUC8 (Figure 2B), was isolated from TOS17 and T‐DNA insertional rice mutants . COW1mutants exhibited narrow leaves and a rolled leaf phenotype, which is likely attributable to insufficient water supply due to the small root‐to‐shoot ratio. Fujino et al.  identified another allele of COW1, narrowleaf7 (NAL7). The NAL7 mutant shows a similar but milder phenotype compared with COW1, and the IAA content in NAL7 was reduced compared to the wild type. In addition, overexpression of NAL7 cDNA gave rise to overgrowth and abnormal morphology of the root, which was likely attributable to the overproduction of auxin. These results suggested that NAL7/OsYUC8 is also involved in auxin biosynthesis. The importance of TAA gene in IAA biosynthesis in rice was demonstrated by fishbone (FIB) mutant . FIB exhibited pleiotropic abnormal phenotypes including dwarfing, narrow and adaxially rolled leaves with large lamina joint angles, abnormal vascular development, and lack of crown and lateral roots. In addition, FIB also showed lack of gravitropism and aberrant phyllotaxy deviated from the normal distichous one. Map‐based cloning revealed that FIB encodes co‐ortholog of TAA1 in rice (Figure 2A). Interestingly, loss‐of‐function of FIB resulted in not only the reduction in IAA level but also higher sensitivity to IAA and lower PAT activity. These results suggest that auxin biosynthesis, transport, and sensitivity are interrelated, which might be attributable to the pleiotropic abnormal phenotypes of FIB . Rice genome includes 2 and 14 genes belong to the TAA and YUC families, respectively (Figure 2) . While the inactivation of a single TAA or YUC gene showed no obvious defects in arabidopsis, distinct abnormal phenotypes were appeared in FIB or COW1/NAL7 mutants in rice, suggesting that functional redundancy among TAA or YUC genes is less prevalent in rice than in Arabidopsis.
In contrast, rice narrowleaf1 (NAL1) encodes a trypsin‐like serine and cysteine protease, whose relationship between auxin remains unknown, but NAL1 mutant showed narrow leaves, dwarfing, and defective vascular patterns together with reduced PAT activity . Surprisingly, several agronomic QTLs involved in flag leaf width (qFLW4; , WFL; ), photosynthesis rate (GPS; ), flag leaf shape (qLSCHL4; ), and spikelet number (SPIKE; ) were allelic to NAL1. The increased yield in indica rice varieties, which introduced these QTLs, suggests that NAL1 is available in plant breeding. The latest study uncovered that NAL1 functions in the regulation of cell division during leaf primordia initiation . In NAL1 mutant, expression of several G1‐ and S‐phase specific genes were reduced, suggesting that NAL1 affects cell‐cycle regulation. In addition, the reduced expressions were also shown in PIN1, three auxinresponsefactorARF genes, and three YAB genes, but the expression of YUC genes were comparable to those of wild type. These results indicated that the inactivation of NAL1 affects auxin transport and auxin response but not auxin biosynthesis .
Overall, auxin‐related narrow leaf mutants exhibit pleiotropic abnormal phenotypes other than the reduction in leaf width. The representative phenotypes seem to be appeared in vascular patterning and root growth since auxin plays critical role in the development of these organs.
3. CSLD‐related narrow leaf mutants
Cell walls are essential structures surrounding plant cells. While cells are expanding, primary cell walls fulfill the support and barrier functions. After cell expansions are completed, secondary cell walls are formed between primary walls and plasma membranes, giving additional strength to cells. Cell wall is composed of polysaccharides, proteins, and phenolic compounds. Classically, polysaccharides are classified into cellulose, hemicelluloses, and pectins . Cellulose synthase (CesA) protein contains a zinc finger domain at the N‐terminus, eight transmembrane domains, and a central catalytic domain known as “D_D_D_QxxRW” motif. Although the mechanism by which CesA creates β‐1,4‐glucan chain is not fully revealed, it is plausible that glucan chain synthesized by the catalytic domain in the cytoplasm goes out of plasma membrane through the pores formed by the transmembrane domain . It is likely that the zinc finger domain at the N‐terminus is involved in CesA protein dimerization, leading to the higher‐order structures [58, 59].
Based on the sequence similarity to CesA genes, a large superfamily of at least 41 cellulosesynthase‐like (CSL) genes were found in the Arabidopsisthaliana genome . They were classified into six subfamilies (CSLA, B, C, D, E, and G), and subsequent studies identified three additional CSL subfamilies (CSLF, H, and J) [61, 62]. CSL proteins contain sequence motifs that are characteristics of β‐glycosyltransferases. The only difference of CSLs from CesAs is the lack of the zinc finger domains at the N‐terminus, which seems to be particularly important to form higher‐order structures. In addition, most CSL proteins appear to be localized not in the plasma membrane but in the Golgi, where hemicellulose synthesis takes place. From these characteristics, CSL genes are predicted to catalyze the biosynthesis of noncellulosic polysaccharides . As far as we know, the first biochemical evidence was provided by the soybean somatic embryos, in which expression of guar CSLA candidate cDNA gave rise to the enhanced mannan synthase activity . Subsequent studies demonstrated that the CSLA genes encode (gluco)mannan synthases [64, 65], and that the CSLF and CSLH genes encode mixed linkage glucan synthases [66, 67]. CSLC genes were predicted to be involved in the xyloglucan synthesis , but recent study reported that some CSLC genes of barley are targeted to the plasma membrane, suggesting that the CSLC subfamily contains more than one type of polysaccharide synthase .
The uneven distribution of CSL genes implies how CSL subfamilies have been evolved in parallel with the diversification of plant species. While CSLB and CSLG subfamilies are found only in eudicots, CSLF, CSLH, and CSLJ subfamilies are specific to Poaceae. Particularly, CSLJ subfamily is unique in that it is only found in certain grasses, such as barley, wheat, sorghum, and maize, but not in rice or Brachypodium . In contrast, CSLD subfamily is commonly found in all land plants, and show the highest similarity to CesA family among CSL subfamilies at sequence levels. The small number of introns and the gene structure diversity within the subfamily imply the possibility that CSLD is the oldest gene family in the cellulose synthase superfamily [60, 70]. Genome database survey revealed that CSLD subfamily contains six Arabidopsis genes, five maize genes, five rice genes, three barley genes, five sorghum genes, and six Brachypodium genes, and subsequent phylogenetic analysis showed that they are further classified into three clades (Figure 3) [71, 72]. The first clade including AtCSLD1 and AtCSLD4 is specifically expressed in pollens and involved in pollen tube elongation , and the second clade including AtCSLD2, AtCSLD3, OsCSLD1, and ZmCSLD5 is highly expressed in root tissues and involved in root hair development [73–77]. While these two clades are commonly involved in “tip‐growing” development, the loss‐of‐function of the third clade including AtCSLD5, OsCSLD4, and ZmCSLD1 exhibited different phenotypes.
In rice, inactivation of OsCSLD4 resulted in distinct narrow leaf phenotype. So far, several narrow leaf genes such as narrowleafanddwarf1 , narrowandrolledleaf1 , Oscd1 , slenderleaf1 , dwarfandnarrowedleaf1 , and dwarfandnarrowleaf3  were allelic to OsCSLD4. The mutants commonly exhibited narrow and rolled leaves and dwarfing phenotypes. The reduction in leaf‐blade width and plant height was clearly attributable to the decrease of cell number, suggesting that OsCSLD4 promotes cell proliferation activity. But if so, why is leaf‐blade length less affected by the mutation than that of leaf‐blade width? This question was solved by the increase of cell length in OsCSLD4 mutant. Plants are able to compensate for a reduction in cell number with an increase in cell size , and the degree of compensation may differ depending on the direction. In fact, the number of cells was equally reduced in both length and width direction in OsCSLD4 . The expression analysis revealed that OsCSLD4 is specifically expressed in M‐phase cells in all developing organs, and loss‐of‐function of OsCSLD4 resulted in the alteration of cell‐cycle regulation. Interestingly, OsCSLD4 included cells with 4C nucleus while such cells were not detected in normal rice. These results suggested that OsCSLD4 plays a pivotal role in M‐phase to progress cell proliferation .
The inactivation of ZmCSLD1 also results in the narrow leaf and fine stem phenotype mainly due to the decrease of cell number . In addition, wart‐like cell clusters were formed on the leaf surface. The warts were attributable to the defects of cell division in leaf development, and disrupted cross‐wall formations were frequently observed in epidermal cells. Such defective developments of cell wall often appeared in cytokinetic mutants of Arabidopsis, such as knolle , korrigan , and hinkel , in which impairment of cytokinesis was caused by a failure of cell‐plate formation. Considering the nature of CSLD as a wall‐synthesizing enzyme, the M‐phase specific expression, and the defective cell wall development in the mutant, it is speculated that CSLD may be involved in cell‐plate formation. The existence of CSLD genes in all land plants also suggests the fundamental function of this subfamily. Recently, it was revealed that transiently expressed AtCSLD5 is involved in mannan synthesis in tobacco leaves . Distantly related CSLA subfamily also exhibits mannan synthase activity, but CSLA proteins readily use Guanosine diphosphate (GDP)-glucose as well as GDP‐mannose and hence efficiently synthesize glucomannans [64, 88]. Since the mannosyltransferase activity of AtCSLD5 was reduced by adding GDP‐glucose together with GDP‐mannose, CSLD subfamily is involved in a different kind of mannan synthesis from that catalyzed by CSLA subfamily . Although mannans have been well studied as storage component, little information has been accumulated in relation to cytokinesis. Further analysis will reveal the detailed mechanism of plant cytokinesis and novel functions of hemicelluloses.
Overall, CSLD‐related narrow leaf mutants exhibit a decrease in the whole plant size other than the reduction in leaf width. These phenotypes are directly attributable to the reduced cell proliferation activity, for CSLDs of the third clade are predicted to fulfill a function closely related to cytokinesis.
4. Polarity‐related narrow leaf mutants
Most plant leaves are asymmetrical in all directions. Grass family leaves include leaf‐blade in the distal side, leaf‐sheath in the proximal side, and lamina‐joint between the leaf‐blade and leaf‐sheath. The bulliform cells, which curl leaf‐blades to prevent over transpiration, and xylems are formed only on the adaxial side, and the phloems on the abaxial side. The midrib, which functions as a physical support for the leaves, and ligule are formed in the medial side, and the sawtooth hairs and auricle in the lateral side (Figure 1A–E) . For the construction of such a sophisticated organ, the proximal‐distal, adaxial‐abaxial, and medial‐lateral polarities must be constructed as soon as cells acquire leaf fate in SAM (Figures 1F and G).
Among the three polarities, the molecular mechanism of adaxial‐abaxial polarity is well studied using Arabidopsis. Through the loss‐of‐function and/or gain‐of‐function analyses, it has been revealed that adaxial identity is regulated by class III homeodomain‐leucine zipper (HD‐Zip) family genes and asymmetricleaves2 (AS2), and that abaxial identity by yabby (YAB) family genes, kanadi (KAN) family genes, and auxinresponsefactor (ARF) family genes . The adaxial or abaxial specific expression of these genes is crucial for the establishment of the organ polarity, and these regulators are interacting antagonistically [17, 90]. In addition, small RNAs are also involved in the negative regulation of these regulators to maintain the expression regions [90–92]. In rice, the loss‐of‐function of shallot‐like1 (SLL1)/rolledleaf9 (RL9), which encodes SHAQKYF class MYB transcription factor belonging to the KAN family, resulted in the suppression of abaxial development while enhanced expression led to the abaxialized leaf phenotypes [93, 94]. Moreover, in maize, the accumulation of miR166, which is involved in the cleavage of class III HD‐Zip transcripts, defined the expression region of the rolledleaf1 (RLD1) belonging to the class III HD‐Zip family, promoting the establishment of adaxial‐abaxial polarity . Despite the morphological differences from dicots, these genes homologous to KAN or class III HD‐Zip seem to fulfill similar regulation in grass family.
While detail genetic regulators of proximal‐distal polarity remain unclear in Arabidopsis, morphological, and molecular analyses are proceeding in grass families for the convenience of distinct organ development along the proximal‐distal axis. A number of dominant mutations which specifically affect proximal‐distal patterning have been characterized in maize . The dominant mutant Knotted1 (Kn1) was characterized by sheath‐like cells in the leaf‐blade . KN1 encodes a homeodomain protein, and KN1 transcripts were localized in the meristem but excluded from the leaf initial cells [98, 99]. However, KN1 proteins were detected outside of the KN1‐transcript localized area, suggesting the noncell‐autonomous nature of KN1 gene . In leaf primordia, KN1 proteins were accumulated in the most proximal part, and ectopic expression of KN1 in the distal leaf‐blade gave rise to alteration into sheath cell identity [101, 102]. These results suggested that KN1 is involved in the establishment of proximal identity in leaf development. Ectopic expressions of KN1‐likehomeobox (KNOX) genes also resulted in cell fate alterations in maize, barley, and Arabidopsis, suggesting the highly conserved function of KNOX genes [103–105]. On the other hand, PIN1 proteins which mediate polar auxin transport (PAT) are highly expressed in the distal ends of developing leaf primordia. Auxin plays pivotal roles in leaf development as we mentioned above, and PIN1 creates an auxin maximum in the distal end of leaf primordium [31, 106]. The subsequent canalization through the interior of leaf primordia leads to the development of primary vascular strand. Thus, the auxin gradient along the proximal‐distal axis is likely to play pivotal role in leaf development. Maize liguleless1 (LG1) and liguleless2 (LG2) mutants lack both ligule and auricle between leaf‐blade and leaf‐sheath [107–109]. It was revealed that LG1 encodes a squamosa‐promoter binding protein, and that LG2 encodes a basic leucine zipper protein [110, 111]. While LG1 is specifically expressed in ligule initiating area, LG2 shows earlier and broader expression pattern than that of LG1 [112, 113]. The phenotype of lg1lg2 double mutant suggested that they act in the same pathway, implying the possibility of interaction between LG1 and LG2 . In addition, other liguleless mutants have been identified such as LG3 and LG4, which encode class I KNOX genes [114, 115]. These findings promote the construction of a hypothetical model of leaf‐blade‐sheath boundary formation .
Compared with other polarities, the molecular mechanism of the medial‐lateral polarity is less understood. So far, it was revealed that droopingleaf (DL) plays pivotal role in the development of medial organs in rice. DL encodes a putative transcription factor belonging to the YAB family, and DL mutants showed defective development of a midrib in the leaf, leading to the drooped leaf phenotype . The DL transcripts were localized in the central region of leaf primordia, and over‐expression of DL resulted in the ectopic formation of midrib‐like structures in the lateral regions as well as in the central region of the leaf. In contrast, the development of leaf lateral domains is highly regulated by wuschel‐relatedhomeobox (WOX) genes. In maize, the loss‐of‐function mutations in both narrowsheath1 (NS1) and NS2 resulted in the significant reduction in leaf width due to the lack of marginal regions in leaves [117–119]. NS1 andNS2 double mutants fail to downregulate KNOX proteins in the premarginal regions of leaf primordia, leading to the deletion of marginal region from the primordial stages [117, 119]. NS1 and NS2 encode the duplicated WOX3 genes, and NS transcripts are accumulated in the marginal edges of initiating leaf primordia. From these results, it was suggested that NS genes play pivotal roles in the recruitment of leaf founder‐cells by downregulating KNOX accumulation [120–122]. Genes belonging to WOX3 family are largely classified into two clades (Figure 4), and the NS‐related clade includes narrowleaf2 (NAL2) and NAL3 of rice, narrowleafeddwarf1 (NLD1) of barley, and pressedflower1 (PRS1) of Arabidopsis .
The nucleotide sequences of NAL2 and NAL3 are identical, corresponding with the recent duplication of a large chromosomal segment in chromosomes 11 and 12 . NAL2/3 and NLD1 mutants show the similar abnormal phenotypes to NS1 and NS2 such as distinct narrow leaf phenotype and defective marginal development, which are attributable to the lack of marginal regions (Figure 5) [123, 125, 126]. The expression patterns of NAL2/3 and NLD1 are also similar to that of NS1NS2, suggesting the conserved function of NS‐related genes in the development of lateral organs. Interestingly, no distinct abnormal phenotypes were observed in the leaf of PRS1 mutant except for the deletion of the proximal lateral stipules . This result supported the leaf‐zonation model that the lower leaf zone of bifacial monocot leaves corresponds with the basal part of bifacial eudicot leaves including stipules . It is, therefore, considered that NS‐related WOX3 genes are involved in the development of the lateral domain in the lower leaf zone.
The width of PRS leaves was significantly reduced by the additional mutation of WOX1. WOX1 is unique in that it belongs to the same clade of the WOX3/PRS family but seems to be absent in grasses (Figure 4) [128, 129]. WOX1 and PRS double mutants exhibit not only the loss of leaf marginal tissues but also the confused adaxial‐abaxial identity at leaf marginal regions [129, 130]. These results suggested that leaf margin functions as an adaxial‐abaxial boundary, where adaxial and abaxial regulators are downregulated by WOX genes .
While the medial‐lateral polarity is directly related to leaf width, mutation or over‐expression of the genes regulating the proximal‐distal or adaxial‐abaxial polarity can also result in the reduction in leaf width together with the alteration of organ polarities. Recessive mutant roughsheath2 (RS2) of maize exhibits narrow/bladeless leaves with a disruption of the blade‐sheath boundary . In RS2 mutant, class I KNOX proteins are ectopically accumulated, and it was revealed that RS2 encodes an MYB‐domain protein, an ortholog of AS1 in Arabidopsis. Thus, it is likely that RS2 is involved in the proximal‐distal patterning by downregulating KNOX expression. Liguleless (LG) genes play pivotal role in the establishment of the boundary between leaf‐blade and leaf‐sheath in the proximal‐distal axis. Recently, another LG gene ligulelessnarrow (LGN) was identified, and its semidominant mutant (LGN‐R) showed narrow leaves with greatly reduced auricle and ligule and indefinite blade‐sheath boundary . LGN encodes a grass‐specific kinase, which is broadly expressed in maize organ but affects LG1 and LG2 expression. The dominant mutant Wavyauricleinblade1 (WAB1) shows narrow leaves with ectopic auricle and extended sheath in leaf‐blade [112, 133]. In contrast to LGN‐R, LG1 was misexpressed in WAB1, and recently it was revealed that WAB1 encodes a teosinte-branched1/cycloidea/PCF (TCP) transcription factor, which is necessary for LG1 expression . These genes play a pivotal role in the establishment of proximal‐distal polarity, but affect leaf width indirectly. Thus, it was considered that proximal‐distal patterning may link to medial‐lateral growth.
On the other hand, Rice SLL1/RL9 encodes KAN transcription factor as we mentioned above, and sll1/rl9 mutants show rolled leaf phenotypes due to the defective development of the sclerenchymatous cells on the abaxial side together with the reduction in leaf width [93, 94]. Similar defective development was observed in semi‐rolledleaf2 (SRL2), which exhibits narrow incurved leaves due to the defective development of sclerenchymatous cells on the abaxial side . SRL2 encodes a novel plant‐specific protein of unknown biochemical function, and highly expressed in the abaxial cell layer in the leaf sheath. However, SLL1/RL9 expression was unaffected in SRL2, and SRL2SLL1 double mutants showed more severe defective development of sclerenchymatous cells on the abaxial side together with the much narrower leaf phenotype than single mutants . These results suggest that SLL1/RL9 and SRL2 function in distinct pathways to regulate the abaxial development. Overexpression of OsHOX32, a member of class III HD‐Zip family, resulted in narrow and adaxially rolled leaves due to the reduction in bulliform cell number . Among the six OsYAB genes, OsYAB1, OsYAB2, and OsYAB6 were upregulated while OsYAB3, OsYAB4, and OsYAB5 were downregulated in the overexpression plants, suggesting the direct or indirect interaction between OsHOX32 and OsYAB genes. Similar defective development was observed by the overexpression of OsLBD3‐7, which shows high similarity to AS2 of Arabidopsis. OsLBD3‐7 overexpression plants exhibit narrow and adaxially rolled leaves due to the reduction in bulliform cell size and number . Since the negative regulators of bulliform cell development were upregulated in overexpression plants, it was suggested that OsLBD3‐7 positively regulate these negative regulators in leaf development. The marginal expressions of NS genes are disappeared in maize raggedseedling2 (RGD2) mutant, which exhibits thread‐like narrow leaves . RGD2 encodes argonaute7 (AGO7)‐like protein, which is involved in the synthesis of trans‐acting short‐interfering RNA (ta‐siRNA) derived from TAS3 in Arabidopsis. So far, several mutants for TAS3 ta‐siRNA pathway have been identified including AGO7‐related genes (RGD2 in maize; , shootless4 (SHL4)/shootorganization2 (SHO2) in rice; ), SGS3‐related gene (leafbladeless1 [LBL1] in maize; [140, 141]), RDR6‐related gene (SHL2 in rice; [142, 143]), and DCL4‐related gene (SHO1 in rice; [143, 144]). Although maize and rice leaves are different morphologically, the loss‐of‐function of these genes commonly gave rise to thread‐like narrow leaves which showed defective adaxial‐abaxial and medial‐lateral polarities. TAS3 ta‐siRNA is expressed on the adaxial side of developing leaf primordia and restricts the expression region of abaxial factor ARF3a and miR166 . Since miR166 restricts the expression region of class III HD‐Zip genes, inactivation of TAS3 ta‐siRNA pathway results in the upregulation of ARF3a and miR166, and downregulation of class III HD‐Zip genes, leading to the abaxialization of leaf. Such a severe abaxialization might disturb the establishment of medial‐lateral polarity. In Arabidopsis, triple mutation of YAB genes (FILYAB3YAB5) has resulted in the thread‐like narrow leaves which showed defective adaxial‐abaxial and medial‐lateral polarities [145, 146]. These results suggest that the establishment and/or development of the medial‐lateral polarity is regulated downstream of the adaxial‐abaxial polarity.
Overall, polarity‐related narrow leaf mutants exhibit distinct reduction in leaf‐blade width together with the disruption of organ polarity. The loss‐of‐function of lateral identity is directly reflected in the reduction of leaf width, but the disruption of the proximal‐distal or adaxial‐abaxial polarities also affect the establishment or development along medial‐lateral axis, suggesting the interactive development between the three polarities.
The reduction in leaf width is a subtle morphological alteration, but the analyses of narrow leaf mutants have uncovered molecular functional diversity of the causal genes. Through a variety of genetic approaches, it has been demonstrated that NS‐related WOX3 genes are critical for the development of leaf lateral domains. Although NS‐related WOX3 transcripts are strictly limited within the marginal edges, the phenotypic alteration of loss‐of‐function mutants occurs in more broad area, suggesting the noncell‐autonomous nature of NS‐related WOX3 genes. This could be explained by the migration of either WOX3 protein itself or the secondary signals derived from the marginal cells. Recently, it was reported that barley NLD1 mutant exhibited malformation of commissural veins in the leaf lateral domain . Since polar auxin transport plays an important role in determining vascular pattern in leaves, nld1 may include some abnormalities in auxin transport. Therefore, it is quite interesting whether auxin functions as the secondary signal of NS‐related WOX3 genes. Auxin plays pleiotropic role in plant development, and at the cellular level, auxin regulates cell division, cell elongation, and cell differentiation. In addition, it is suggested that auxin biosynthetic YUC genes are expressed in response to the juxtaposition of adaxial and abaxial domains . Thus, auxin biosynthesis at the adaxial‐abaxial boundary partly contributes to leaf margin expansion, and this might explain the reduction in leaf width attributable to the disruption of the adaxial‐abaxial polarity. At the downstream of these mechanisms, cell proliferation activity is maintained by CSLD genes of the third clade. The details of plant cytokinesis are not fully understood, particularly as to the components of cell plate. All we covered here is just a part of well‐studied mutants, and there should be many hither‐to unidentified narrow leaf mutants. Further study will give us a novel and detailed mechanism of leaf development in the grass family.
6. Materials and methods
6.1. Plant materials
For morphological observation of barley shoot, a wild type line Kanto Nijo 29 (KN29), which has two‐rowed spike and covered caryopsis, and its gamma‐ray induced narrowleafeddwarf1 (NLD1) mutant, NLD1.b, were used. To promote germination, seeds were kept at 15°C on wet paper for 3 days. Then, imbibed seeds were sown on soil and grown under natural conditions.
6.2. Paraffin sectioning and histological analysis
Plant samples were fixed with FAA (formaldehyde:glacial acetic acid:50% ethanol [2:1:17]) for 24 h at 4°C for histological analysis. They were then dehydrated in a graded ethanol series, substituted with 1‐butanol, and embedded in Paraplast® Plus (McCormick Scientific). The samples were sectioned at 8 μm thickness using a rotary microtome. For the histological analysis, sections were stained in hematoxylin or double‐stained in safranin and fast green. After staining, sections were mounted with Poly‐Mount® (Polysciences, Inc.) and observed with a light microscope.
6.3. Epidermal cell observation
The leaf‐blades were fixed with FAA (formaldehyde:glacial acetic acid:50% ethanol [2:1:17]) for 24h at 4°C. They were then dehydrated in a graded ethanol series. Dehydrated samples were incubated at 96°C in chloralhydrate dissolved in 100% ethanol until they were cleared, and observed with a light microscope.
6.4. Phylogenetic analysis
For the phylogenetic analysis of TAA‐, YUCCA‐, CSLD‐, and WOX3‐related genes, amino acid sequences were obtained from TIGR (