A detailed analysis of the leaf rolling mutant sll2 reveals complex nature in regulation of bulliform cell development in rice (Oryza sativa L.)

Bulliform cells are large, thin‐walled and highly vacuolated cells, and play an important role in controlling leaf rolling in response to drought and high temperature. However, the molecular mechanisms regulating bulliform cell development have not been well documented. Here, we report isolation and characterisation of a rice leaf‐rolling mutant, named shallot‐like 2 (sll2). The sll2 plants exhibit adaxially rolled leaves, starting from the sixth leaf stage, accompanied by increased photosynthesis and reduced plant height and tiller number. Histological analyses showed shrinkage of bulliform cells, resulting in inward‐curved leaves. The mutant is recessive and revertible at a rate of 9%. The leaf rolling is caused by a T‐DNA insertion. Cloning of the insertion using TAIL‐PCR revealed that the T‐DNA was inserted in the promoter region of LOC_Os07 g38664. Unexpectedly, the enhanced expression of LOC_Os07 g38664 by the 35S enhancer in the T‐DNA is not responsible for the leaf rolling phenotype. Further, the enhancer also exerted a long‐distance effect, including up‐regulation of several bulliform cell‐related genes. sll2 suppressed the outward leaf rolling of oul1 in the sll2oul1 double mutant. We conclude that leaf rolling in sll2 could be a result of the combined effect of multi‐genes, implying a complex network in regulation of bulliform cell development.     

Further insight into the role of <Emphasis Type="Italic">KAN1</Emphasis>, a member of <Emphasis Type="Italic">KANADI</Emphasis> transcription factor family in rice

The rice EMS-derived mutant leaf adaxialized 1 (lad1) was isolated based on its upward rolling leaf phenotype. Besides the adaxially rolled leaf, many other agronomic traits were also compromised in lad1. The rolling trait was characterized by a noticeable alteration of bulliform cells in the adaxial side of the leaves. Map-based cloning showed a single nucleotide substitution in the promoter region of the KAN1 gene in lad1 mutant. Further, over-expressing and CRISPR/cas9-edited knockdown transgenic plants confirmed that KAN1 was responsible for the mutant phenotype of lad1. Yeast two-hybrid and bimolecular fluorescence complementation assay demonstrated that KAN1 can interact with the auxin response factors ARF3, ARF7 and ARF15. Physiologically, the contents of auxin (IAA), abscisic acid (ABA), jasmonic acid (JA) and gibberellin (GA) were all significantly increased in the lad1 mutant. Moreover, the GA3 content dramatically decrease in wild-type, but increased in lad1 under IAA induction. Additionally, the expression levels of several IAA and GA biosynthesis and responsive-related genes and genes involved in leaf polarity determination were altered in lad1. Therefore, we hypothesized that KAN1/ARFs protein complexes act as auxin-dependent regulatory units that play a conserved role in leaf development.     

CLD1/SRL1 modulates leaf rolling by affecting cell wall formation,epidermis integrity and water homeostasis in rice

Leaf rolling is considered as one of the most important agronomic traits in rice breeding. It has been previously reported that SEMI‐ROLLED LEAF 1 (SRL1) modulates leaf rolling by regulating the formation of bulliform cells in rice (Oryza sativa); however, the regulatory mechanism underlying SRL1 has yet to be further elucidated. Here, we report the functional characterization of a novel leaf‐rolling mutant, curled leaf and dwarf 1 (cld1), with multiple morphological defects. Map‐based cloning revealed that CLD1 is allelic with SRL1, and loses function in cld1 through DNA methylation. CLD1/SRL1 encodes a glycophosphatidylinositol (GPI)‐anchored membrane protein that modulates leaf rolling and other aspects of rice growth and development. The cld1 mutant exhibits significant decreases in cellulose and lignin contents in secondary cell walls of leaves, indicating that the loss of function of CLD1/SRL1 affects cell wall formation. Furthermore, the loss of CLD1/SRL1 function leads to defective leaf epidermis such as bulliform‐like epidermal cells. The defects in leaf epidermis decrease the water‐retaining capacity and lead to water deficits in cld1 leaves, which contribute to the main cause of leaf rolling. As a result of the more rapid water loss and lower water content in leaves, cld1 exhibits reduced drought tolerance. Accordingly, the loss of CLD1/SRL1 function causes abnormal expression of genes and proteins associated with cell wall formation, cuticle development and water stress. Taken together, these findings suggest that the functional roles of CLD1/SRL1 in leaf‐rolling regulation are closely related to the maintenance of cell wall formation, epidermal integrity and water homeostasis.     

ESCRT-III component OsSNF7.2 modulates leaf rolling by trafficking and endosomal degradation of auxin biosynthetic enzyme OsYUC8 in rice

The endosomal sorting complex required for transport (ESCRT) is highly conserved in eukaryotic cells and plays an essential role in the biogenesis of multivesicular bodies and cargo degradation to the plant vacuole or lysosomes. Although ESCRT components affect a variety of plant growth and development processes, their impact on leaf development is rarely reported. Here, we found that OsSNF7.2, an ESCRT-III component, controls leaf rolling in rice (Oryza sativa). The Ossnf7.2 mutant rolled leaf 17 (rl17) has adaxially rolled leaves due to the decreased number and size of the bulliform cells. OsSNF7.2 is expressed ubiquitously in all tissues, and its protein is localized in the endosomal compartments. OsSNF7.2 homologs, including OsSNF7, OsSNF7.3, and OsSNF7.4, can physically interact with OsSNF7.2, but their single mutation did not result in leaf rolling. Other ESCRT complex subunits, namely OsVPS20, OsVPS24, and OsBRO1, also interact with OsSNF7.2. Further assays revealed that OsSNF7.2 interacts with OsYUC8 and aids its vacuolar degradation. Both Osyuc8 and rl17 Osyuc8 showed rolled leaves, indicating that OsYUC8 and OsSNF7.2 function in the same pathway, conferring leaf development. This study reveals a new biological function for the ESCRT-III components, and provides new insights into the molecular mechanisms underlying leaf rolling.     

Identification of a novel candidate gene for rolled leaf in rice

A semi-narrow and adaxially rolled leaf mutant, rl15(t), was induced from Korean japonica rice cultivar Ilpum by chemical mutagenesis using ethyl methanesulfonate. We characterized the mutant and identified the novel gene causing the mutant phenotype. Cytological analysis of mutant leaves indicated that the adaxial leaf-rolling phenotype is due to the reduced size and number of bulliform cells in the mutant. Genetic analysis showed that the rolled leaf trait is controlled by a single recessive gene, designated rl15(t). Using an F₂ mapping population generated from a cross between Milyang23 and the mutant, we mapped the candidate region to a 174 kb interval on the long arm of chromosome 1 near the centromeric region. Through whole genome sequencing in bulk and MutMap analysis, we identified the causal SNP within the candidate region. The results of RT-PCR analysis indicated that a splicing error occurred due to a base change from G to A at the beginning of the fifth intron of LOC_Os01g37837, which encodes a putative seryl-tRNA synthetase, resulting in the mutant phenotype. Further study of the rl15(t) gene will facilitate analysis of leaf architecture and morphogenesis in rice plants.     

一份水稻叶片反卷突变体的遗传分析及电镜显微观察

叶片是植物进行光合作用的重要器官。叶片适度卷曲能够提高水稻(Oryza sativa)生长中后期群体基部的光能利用率, 因而有利于水稻产量的提高。该研究首先在水稻T-DNA插入突变体库中发现一份叶片反卷的突变体。遗传分析表明, 该性状受到1对隐性核基因控制。扫描电镜观察结果显示, 突变体成熟叶片上下表皮的气孔发生了畸变; 且叶片上表皮气孔数目增多, 而下表皮气孔数目与野生型基本相同。叶片横切面电镜观察结果表明, 与野生型相比, 突变体叶片的泡状细胞数目和面积在早期(二叶期)就开始增加, 在成熟期更加明显, 这可能是导致叶片反卷的主要原因。     

Identification and characterization of NARROW AND ROLLED LEAF 1, a novel gene regulating leaf morphology and plant architecture in rice

Leaf morphology is an important agronomic trait in rice breeding. We isolated three allelic mutants of NARROW AND ROLLED LEAF 1 (nrl1) which showed phenotypes of reduced leaf width and semi-rolled leaves and different degrees of dwarfism. Microscopic analysis indicated that the nrl1-1 mutant had fewer longitudinal veins and smaller adaxial bulliform cells compared with the wild-type. The NRL1 gene was mapped to the chromosome 12 and encodes the cellulose synthase-like protein D4 (OsCslD4). Sequence analyses revealed single base substitutions in the three allelic mutants. Genetic complementation and over-expression of the OsCslD4 gene confirmed the identity of NRL1. The gene was expressed in all tested organs of rice at the heading stage and expression level was higher in vigorously growing organs, such as roots, sheaths and panicles than in elsewhere. In the mutant leaves, however, the expression level was lower than that in the wild-type. We conclude that OsCslD4 encoded by NRL1 plays a critical role in leaf morphogenesis and vegetative development in rice.     

Rolling-leaf14 is a 2OG-Fe (II) oxygenase family protein that modulates rice leaf rolling by affecting secondary cell wall formation in leaves

As an important agronomic trait, leaf rolling in rice (Oryza sativa L.) has attracted much attention from plant biologists and breeders. Moderate leaf rolling increases the amount of photosynthesis in cultivars and hence raises grain yield. Here, we describe the map-based cloning of the gene RL14, which was found to encode a 2OG-Fe (II) oxygenase of unknown function. rl14 mutant plants had incurved leaves because of the shrinkage of bulliform cells on the adaxial side. In addition, rl14 mutant plants displayed smaller stomatal complexes and decreased transpiration rates, as compared with the wild type. Defective development could be rescued functionally by the expression of wild-type RL14. RL14 was transcribed in sclerenchymatous cells in leaves that remained wrapped inside the sheath. In mature leaves, RL14 accumulated mainly in the mesophyll cells that surround the vasculature. Expression of genes related to secondary cell wall formation was affected in rl14-1 mutants, and cellulose and lignin content were altered in rl14-1 leaves. These results reveal that the RL14 gene affects water transport in leaves by affecting the composition of the secondary cell wall. This change in water transport results in water deficiency, which is the major reason for the abnormal shape of the bulliform cells.     

The ADAXIALIZED LEAF1 gene functions in leaf and embryonic pattern formation in rice

The adaxial-abaxial axis in leaf primordia is thought to be established first and is necessary for the expansion of the leaf lamina along the mediolateral axis. To understand axis information in leaf development, we isolated the adaxialized leaf1 (adl1) mutant in rice, which forms abaxially rolled leaves. adl1 leaves are covered with bulliform-like cells, which are normally distributed only on the adaxial surface. An adl1 double mutant with the adaxially snowy leaf mutant, which has albino cells that specifically appear in the abaxial mesophyll tissue, indicated that adl1 leaves show adaxialization in both epidermal and mesophyll tissues. The expression of HD-ZIPIII genes in adl1 mutant increased in mature leaves, but not in the young primordia or the SAM. This indicated that ADL1 may not be directly involved in determining initial leaf polarity, but rather is associated with the maintenance of axis information. ADL1 encodes a plant-specific calpain-like cysteine proteinase orthologous to maize DEFECTIVE KERNEL1. Furthermore, we identified intermediate and strong alleles of the adl1 mutant that generate shootless embryos and globular-arrested embryos with aleurone layer loss, respectively. We propose that ADL1 plays an important role in pattern formation of the leaf and embryo by promoting proper epidermal development.     

Post-Golgi trafficking of rice storage proteins requires the small GTPase Rab7 activation complex MON1–CCZ1

Protein storage vacuoles (PSVs) are unique organelles that accumulate storage proteins in plant seeds. Although morphological evidence points to the existence of multiple PSV-trafficking pathways for storage protein targeting, the molecular mechanisms that regulate these processes remain mostly unknown. Here, we report the functional characterization of the rice (Oryza sativa) glutelin precursor accumulation7 (gpa7) mutant, which over-accumulates 57-kDa glutelin precursors in dry seeds. Cytological and immunocytochemistry studies revealed that the gpa7 mutant exhibits abnormal accumulation of storage prevacuolar compartment-like structures, accompanied by the partial mistargeting of glutelins to the extracellular space. The gpa7 mutant was altered in the CCZ1 locus, which encodes the rice homolog of Arabidopsis (Arabidopsis thaliana) CALCIUM CAFFEINE ZINC SENSITIVITY1a (CCZ1a) and CCZ1b. Biochemical evidence showed that rice CCZ1 interacts with MONENSIN SENSITIVITY1 (MON1) and that these proteins function together as the Rat brain 5 (Rab5) effector and the Rab7 guanine nucleotide exchange factor (GEF). Notably, loss of CCZ1 function promoted the endosomal localization of vacuolar protein sorting-associated protein 9 (VPS9), which is the GEF for Rab5 in plants. Together, our results indicate that the MON1–CCZ1 complex is involved in post-Golgi trafficking of rice storage protein through a Rab5- and Rab7-dependent pathway.

The small GTPases Rab5- and Rab7-dependent pathway is involved in rice storage protein trafficking to vacuoles.     

水稻窄卷叶突变体nrl7的鉴定与基因定位

叶片的形态是理想株型的重要性状, 叶片适度卷曲能提高水稻(Oryza sativa)群体的光能利用率, 研究控制水稻叶片形态的相关基因能够进一步丰富株型理论。该研究在粳稻品系C275的群体中发现了1株自然变异的窄卷叶突变体nrl7(narrow rolled leaf 7)。与野生型相比, 突变体的叶片变窄且向内卷曲; 该突变体叶片连接中脉的泡状细胞严重变形, 中脉与小叶脉之间的维管束数量均减少至1个。此外, 突变体nrl7的株高、实粒数和实粒重均降低或减少, 分别为野生型的88.46%、69.77%和68.98%, 差异达极显著水平。叶片卷曲导致单叶光合速率减弱, 与野生型相比, 突变体的光合速率降低了17%, 达极显著水平。突变体nrI7叶片的气孔导度、胞间CO₂浓度和蒸腾速率则与野生型相比无明显变化。利用图位克隆的方法将目的基因定位于水稻第3染色体短臂上的分子标记RM5444和MM1300之间, 物理距离约为185.14 kb。研究结果为该基因的克隆和进一步的功能分析奠定了基础。