Introduction

The diversity of plant body elaborates mainly through the post-embryonic development of aerial lateral organs, which are ultimately derived from franks of a group of self-renewal stem cells within shoot apical meristem (SAM). An indeterminate ground cellular state in meristem that allows cells in SAM to either acquire stem cell identity or be recruited into organ primordia was specified by maintaining a high cytokinin and low GA ratio that directly regulated by the KNOTTED-like Homeobox (KNOX) genes ^{1, 2, 3, 4}. Lateral organogenesis involves the recruitment of founder cells and their transition from an indeterminate cellular state to a determinate fate ². Initiation of the leaf organogenesis is marked by the down-regulation of KNOX expression in a subset of cells in the periphery of the SAM and activation of genes including PHAN, BOP and YABBY families that maintain the repression of KNOX expression and the regulation of leaf polarity during leaf development ^{2, 3, 5}. During vegetative developmental stage, the SAM continues to produce leaf primordia in a regular pattern over space (phyllotaxy) and time (plastochron) ⁶. The field inhibitory hypothesis is applauded to explain plant phyllotaxy, proposing that the new primordia will form only after escaping the biochemical constraint made by the existing primordia ⁷. Recent works revealed that the biochemical constraints could be established by polar auxin transport ^{8, 9, 10}. The initiation of lateral organ primordia is induced by a local auxin maximum accumulated in the peripheral zone of SAM, then next primodium formation may initiate at the site most distant to the preexisting primordium because the established primordia act as a sink to deplete auxin accumulation within surrounding cells ^{7, 8, 9, 10}. Since changes in size and/or organization of SAM can alter the field in which auxin acts, a number of mutants exhibit close association of abnormal phyllotaxy with modified meristems ^{11, 12, 13, 14}. The maize abphyl1 mutant has an enlarged SAM with abnormal phyllotaxy ¹³. Molecular analysis of abphyl1 has revealed that ABPHYL1 encodes a cytokinin inducible response regulator, which controls the phyllotactic pattern through negative regulation of the expanding shoot meristems ^{13, 15}. Rice shoot organization (sho) and Arabidopsis altered meristem program 1 (amp1) have malformed SAMs and show irregular patterns of phyllotaxy and plastochron ^{11, 14}. It may have coordination between temporal and spatial regulation of leaf development, because the disruption in phyllotaxy simultaneously affects plastochron in these mutants. It is possible that the inhibition effect of preexisting primodia may not only determine the site of leaf initiation, but also prevent the precocious development of new leaves ^{7, 16}. However, the analysis of the rice plastochron1 mutant that shows decreased plastochron with normal phyllotaxy provided the insight of the disassociation between regulatory pathway governing spatial and temporal patterns of the leaf initiation ^{12, 17}. Molecular analysis revealed that PLA1 encodes CYP78A11, a member of cytochrome P450 family involved in phytohormone biosynthetic pathways ¹⁷. Either exogenous application of gibberellic acid (GA) or inhibition of auxin transport results in changes in phyllotaxy and plastochron, which suggests that phytohormones may regulate both plastochron and phyllotaxy ^{17, 18, 19}.

An alternative mechanism that may regulate the timing and spacing of the leaf initiation comes from the identification of maize TERMINAL EAR1 (TE1), which encodes a putative RNA-binding protein ²⁰. The te1 mutant plant initiates leaves more frequently and exhibits an irregular phyllotaxy in addition to abnormal internode length. Transcripts of TE1 accumulate in a semicircular ring embracing sites of leaf initiation that aligned with leaf midrib ^{20, 21}. The close correlation between the TE1 expression pattern and defects in leaf initiation suggests that the RNA binding protein functions in regulating leaf initiation ^{16, 20}.

Manipulation of plant architecture is thought to be an important way to increase crop yield ^{22, 23}. Isolation of genes regulating plastochron is of agronomical importance, because the number of leaves affects the number of tillers thus determines the number of panicles ^{17, 24}. We here report the characterization of the rice mutant lhd2 and the molecular cloning of the LHD2 gene. The lhd2 mutant exhibits multiple phenotypes including dwarfism, abnormal plastochron, reduced tiller number, and failure of panicle generation, which is similar to the previously identified mutant lhd ^{25, 26}. Molecular analysis showed that LHD2 is a functional homologue of maize TE1. Genome-scale expression profile analysis suggested that LHD2 may play essential roles in regulating plant architecture by interacting with plant hormones and homeobox genes.

Materials and Methods

Plant materials and growth condition

The rice (Oryza sativa L.) lhd2-1 and lhd2-3 were spontaneous mutants of YunDao32 (japonica) and TN1 (indica), and lhd2-2 was isolated from Nipponbare (japonica) mutagenized with ethyl methanesulfonate (EMS). Rice plants were cultivated in the experimental field at the China National Rice Research Institute in Hangzhou in the natural growing seasons.

RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated from 14d wild-type and lhd2 plants by a guanidine thiocyanate extraction method as previously described ²⁷. To conduct RT-PCR analysis, cDNA strands are synthesized as previously described ²². 1.0 g product was subsequently used to amplify the target genes. Primers used in expression pattern analysis were listed in Table 1.

Table 1 - List of the Primers used and molecular markers developed in this study.

Full table

Scanning electron microscopy

Samples were prepared as described previously ²⁸ with some modifications. Briefly, samples were fixed with 2.5% (v/v) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) and incubated at 4 °C overnight. After being rinsed with 0.1 M sodium phosphate buffer, they were post-fixed in 1% (w/v) osmium tetroxide for 2 h at 4 °C and rinsed with the same buffer. Samples were then dehydrated in a graded series of ethanol. For scanning electron microscopy, 100% ethanol was replaced with 3-methylbutyl acetate. Samples were critical-point dried, sputter-coated with platinum, and observed under a scanning electron microscope (model S-570; Hitachi, Tokyo, Japan).

Histological analysis

Samples were fixed with the formalin–acetic acid–alcohol (FAA) fixative solution at 4 °C overnight followed by dehydration steps and then embedded in paraffin (Paraplast Plus, Sigma). The tissues were sliced into 8 mm and dried overnight onto 3-amino-propyltriethoxy silanecoated slides (ProbeOn Plus, Fisher Biotech Co.). Sections were stained with Safranin O and Fast Green (Fisher Biotech Co.) and observed under bright-field through a microscope (Leica DMR) and photographed using a Micro Color Charge-coupled Device (CCD) camera (Apogee Instruments).

Map-based cloning of LHD2

An F₂ mapping population was generated by a cross between the LHD2-1/lhd2-1 heterozygote and MingHui63, a polymorphic indica variety, and the rice genomic DNA was prepared as described ²⁸. The LHD2 locus was diagnostically mapped between SSRs markers RM1361 and RM104 on Chromosome 1 using 36 F₂ plants of lhd2-1 homozygotes, further placed into the DNA fragment between the markers RM37913 and S86300 using 1 080 F₂ lhd2-1 mutant plants, and finally placed in an interval of a 40 kb DNA fragment between the P3 and P5 markers and co-segregated with the P4 marker, which were developed in this work (Table 1). To sequence the lhd2-1, lhd2-2 and lhd2-3 alleles, the entire genomic regions were amplified from each allele and its corresponding wild-type plant by PCR with LA-Taq (TaKaRa, Dalian, China). The mutations in lhd2-1, lhd2-2 and lhd2-3 were identified by directly sequencing the PCR products. To verify the mutation in lhd2 mutant alleles, CAPs1, CAPs2 and P8 (Table 1) were developed for lhd2-1, lhd2-2 and lhd2-3, respectively.

Results

Morphological characterization of the lhd2 mutant

To understand the mechanism underlying the genetic control of plant architecture, we have collected several types of mutants altered in the overall plant body plan. Among them, three mutants showing the similar phenotypes to the leafy-head (lhd) mutants ^{25, 26} were isolated from japonica varieties of Yundao32 and Nipponbare and an indica variety of TN1, respectively. These mutants exhibited dwarfism, shortened plastochron, and a prolonged vegetative developmental stage (Figure 1). Genetic complementation test revealed that the three mutants are allelic (data not shown) and therefore were designated as lhd2-1, lhd2-2 and lhd2-3, respectively. The lhd2-1 mutant was representatively used for the further study in this work.

Figure 1.

Phenotype of the lhd2 mutant plant. (A) 14d seedling of wild type (left) and lhd2-1 (right). (B) 14d seedling of wild type (left) and lhd2-2 (right). (C) 14d seedling of wild type (left) and lhd2-3 (right). (D) Wild-type (left) and lhd2-1 (right) plants at the tillering stage. (E) Wild-type (left) and lhd2-1 (right) plants at the heading stage. (F) and (G) The stems of wild-type and lhd2-1 plants at the heading stage. Bars in A-B = 1 cm, in C = 2 cm, in D–F = 5 cm, and in G = 1 cm.

Full figure and legend (38K)

At the seedling stage, lhd2 plants could be distinguished from the wild type by the rapid emergence of leaves with reduced leaf size and plant height (Figure 1A to 1C). Detailed phenotypic observation revealed that the mutant plants produced less tillers than the wild type at the tillering stage (Figure 1D) and could not form any panicle at the heading stage (Figure 1E).

To find out the role of LHD2, we characterized the lhd2-1 mutant at both anatomical and histological levels. In the longitudinal sections through apices of 14d seedlings, more leaf primordia and tiller buds could be observed in lhd2-1 than that in wild-type plants (Figure 2A, 2D), indicating that the rapid emergence of leaves resulted from the rapid initiation of leaf primordia and the reduced tiller number in the lhd2-1 seedling was a consequence of the suppression of the tiller bud outgrowth other than the defects in tiller bud initiation. Furthermore, the lhd2-1 intercalary meristem was apparently enlarged (Figure 2F) compared with that of the wild type (Figure 2E), producing many more nodes in the lhd2-1 mature plant (Figure 1F and 1G). Nevertheless, the elongation of the internodes was inhibited in lhd2-1 (Figure 1G), which resulted in a dwarf phenotype. Additionally, the lhd2 mutation also affected the phase transition from the vegetative to reproductive growth. Scanning electron microscope (SEM) observation revealed that no significant morphological difference could be found between lhd2-1 and the wild-type SAMs in the seedling apices (Figure 2G and 2H). However, while the wild-type SAM underwent the transition from the vegetative to reproductive phase to generate rachis branches (Figure 2B and 2I), the lhd2 SAM failed in phase transition and maintained the vegetative identity to produce many more leaves than the wild type (Figure 2C and 2J). These results suggest that LHD2 may play multiple roles in the initiation of leaf primordia, elongation of internodes, outgrowth of tiller buds and the phase transition.

Figure 2.

Structure of the lhd2 SAM and stem. (A) Longitudinal section of the14d wild-type shoot apex. (B) Longitudinal section of the wild-type reproductive SAM. (C) Longitudinal section of the lhd2-1 SAM, indicating the block of phase transition from vegetative to reproductive growth. (D) Longitudinal section of the14d lhd2-1 shoot apex. (E) Structure of the 14d wild-type stem in which cells and vascular bundles were randomly arranged, but the node and internode were not differentiated. (F) Structure of the 14d lhd2-1 stem in which the node and internode were clearly differentiated. Arrows in (F) indicate the position of the nodal region. (G) SEM view of the wild-type vegetative SAM. (H) SEM view of the lhd2-1 vegetative SAM. (I) SEM view of the wild-type reproductive SAM. (J) SEM view of the lhd2-1 SAM, indicating the block of the phase transition from vegetative to reproductive growth. Scale bars in (A)-(I) = 100 m, and in (J) = 20 m.

Full figure and legend (52K)

Molecular cloning of LHD2

To map the LHD2 locus, we generated a F₂ mapping population derived from a cross between the LHD2-1/lhd2-1 heterozygote and Minghui63, a polymorphic indica variety. Linkage analysis of 36 F₂ plants that showed the lhd2-1 mutant phenotype primarily placed the LHD2 locus in an interval between SSRs markers RM1361 and RM104 on Chromosome 1 (Figure 3A). To fine-map LHD2, 1 080 F₂ mutant plants were analyzed using 7 newly developed PCR-based markers, P1 to P7 (Table 1 and Figure 3B). LHD2 was pin-pointed within an interval of 40 kb DNA fragment between the P3 and P5 markers and co-segregated with the P4 marker in the BAC clone AP003380. Within this region, there are eight predicted genes, PG1 to PG8. Sequencing these genes in the lhd2-1 allele revealed a G-to-T point mutation at the nucleotide 3 082 in PG5, which leads to a splicing error (Figure 3C). No mutation could be found in the other predicted genes. Mutations were also identified in PG5 in the other lhd2 alleles. In lhd2-2, a single nucleotide C was deleted in the third exon at the nucleotide 1 407, which causes a frameshift and produces a premature translational product (Figure 3C). In lhd2-3, the 1 972 bp retrotransposon was inserted in the third exon at the nucleotide 1 416, which results in a premature translational product. These three mutations were also confirmed by molecular markers developed from the sequences of mutated LHD2 in lhd2-1, lhd2-2 and lhd2-3, respectively (Table 1 and Figure 3D). Moreover, a database search with the LHD2 sequence demonstrated that LHD2 shared the highest sequence similarity to maize TE1 ²⁰ that encodes a protein containing putative RNA recognition motif (RRM) (Figure 4A). Sequence comparison between indica and japonica subspecies revealed only 18 bp differences at the genomic DNA level that caused only 5 changes at the protein level (Figure 4B). Loss-of-function mutation in maize TE1 causes an increase in leaf initiation, an irregular phyllotaxy and an altered internode length ²⁰. Taken all this together, we concluded that LHD2 (accession number: DQ393277), the rice orthorlog of maize TE1, is the gene responsible for the phenotype of lhd2.

Figure 3.

Positional cloning of LHD2. (A) The LHD2 locus was mapped in the Chromosome 1 (Chr 1) between SSRs markers RM1361 and RM104. The numerals indicate the number of recombinants (Rec) identified from 1 080 F2 mutant plants. (B) Fine mapping of the LHD2 locus with the markers (P1 to P7) developed based on the sequence of BAC clone AP003380. The LHD2 locus was narrowed to a 40 kb genomic DNA region between markers P3 and P5 and cosegregated with marker P4. PG1, B1417F08.22; PG2, B1417F08.23; PG3, B1417F08.25; PG4, B1417F08.26; PG5, B1417F08.27; PG6, B1417F08.29; PG7, B1417F08.30; PG8, B1417F08.31. (C) The LHD2 structure, showing the mutated sites of the three lhd2 alleles. The start codon (ATG) and the stop codon (TGA) are indicated. Closed boxes indicate the coding sequence and lines between boxes indicate introns. (D) Molecular identification of the mutations of lhd2-1, lhd2-2 and lhd2-3 by markers CAPs1, CAPs2 and P8, as indicated in Table 1.

Full figure and legend (44K)

Figure 4.

Sequence alignment. (A) Alignment of rice LHD2 and maize TE1. Numbers at left refer to the positions of amino acid and the conserved RRM motifs were underlined. (B) Comparison of the cDNA sequences between subspecies of indica and japonica. Numbers at left refer to the positions of nucleotide. Red letters stand for the polymorphic nucleotides in japonica, green for the polymorphic nucleotides that cause amino acid changes in japonica, and green triangle for the inserted nucleotides in japonica.

Full figure and legend (196K)

The expression pattern of LHD2

To examine the expression pattern of LHD2, we conducted a semi-quantitative RT-PCR analysis using total RNA isolated from different organs. As shown in Figure 5A, LHD2 was expressed mainly in the shoot apex region. No expression could be detected in roots, nodes, internodes, leaves, and leaf sheaths. This organ-specific expression pattern is consistent with the action of LHD2 that is required for the shoot development of rice plant.

Figure 5.

Expression of LHD2 in organs and differentially expressed genes in lhd2-1. (A) The LHD2 expression in organs. Total RNA was isolated from roots (R), nodes (N), internodes (I), leaves (L), leaf sheathes (S) and shoot apices (A) of wild-type plants. (B) Confirmation of differentially expressed genes in the lhd2 mutant plant. Amplification of ubiquitin cDNA was used to ensure that approximately equal amount of cDNA was loaded.

Full figure and legend (31K)

The altered expression patterns of KNOX and phytohormone-related genes in lhd2

To investigate the molecular mechanism of LHD2 in regulating rice plastochron, we generated the genome-scale expression profile between wild-type and lhd2 plants using AFFYMETRIX Rice Gene Chip (Santa Clara, USA). Classification analysis indicated that most of the differentially expressed genes in the mutant plants were related to transcription regulation, signal transduction and hormone response. The representative differential expression genes were summarized in Table 2. Among genes with significant alteration in expression, several genes related to the KNOX signal pathway, cytokinin metabolism and auxin response were confirmed by RT-PCR analysis (Figure 5B). These results suggested that LHD2 may regulate rice leaf initiation and stem elongation through KNOX genes and hormone related genes within the SAM. Further analysis is needed to dissect the regulatory pathway mediated by LHD2 in controlling shoot development of rice plant.

Table 2 - Differentially expressed genes identified by microarray analysis.

Full table

Discussion

The development of leaves, including the position, shape and size, is a major determinant factor that affects plant architecture. Phyllotaxy and plastochron are basic aspects of leaf development and are species specific. Mutants that affect either phyllotaxy or plastochron are useful tools to understand the mechanism of leaf formation. In case of rice, the study of phyllotaxy and plastochron is also of agronomic importance.

In this study, we have identified and characterized the rice mutant lhd2 that exhibits defects in plastochron, stem elongation, tiller bud outgrowth and developmental phase transition. Molecular analysis revealed that LHD2 is homologous to maize TE1 that regulates the rate of leaf initiation in corn plants ²⁰. TE1 displays a significant similarity to Mei2, an RNA-binding protein from Schizosaccharomyces pombe that is required for both premeiotic DNA synthesis and the first reductional division of meiosis ²⁹. The similarity is highest in the three regions in both protein sequences that encode RNA-recognition motifs (RRMs). Previous studies on the sequences similarity demonstrated that this gene shares a much conserved exon-intron structure and a high degree of amino acid similarity among Poaceae species, especially a very high degree identity in conserved RRM motifs ^{20, 21}. Our work showed that the mutation of LHD2 in rice can cause the similar phenotype to maize te1 including shorten plastochron and reduced stem elongation, providing a strong evidence that LHD2 is a functional orthologue of maize TE1. However, unlike the maize te1 mutant that exhibits defects in both plastochron and phyllotaxy, the rice lhd2 mutant exclusively affects plastochron. Additionally, the phase transition from vegetative to reproductive growth is also suppressed in lhd2, indicating the functional diversion of the TE1 family during the process of evolution.

The TE1 expression pattern in Poaceae species revealed by in situ hybridization suggested that TE1 may involve in both leaf initiation and cell differentiation ²¹. Our observation that stem elongation, phase transition and tiller bud outgrowth are all abnormal in lhd2 is consistent with this hypothesis because cell differentiation contributes to all these developmental events. Although molecular genetic and cell biology approaches have made significant advances in understanding the regulatory mechanism in leaf formation, most researches have been focused on phyllotaxy due to the lack of mutants that are exclusively defective in plastochron. The rice PLA1 gene is the first reported gene that is merely involved in regulating plastochron ¹⁷. In view of the different performance between pla1 and lhd2 on tiller number and developmental phase transition, we assumed that LHD2 and PLA1 may regulate rice plant leaf initiation temporally in a distinct genetic pathway.

Field inhibitory theory proposed that existing primordia use either biochemical or biophysical constraints to control phyllotaxy and plastochron ⁵. However, the mechanism regulating plastochron is far to be elucidated. The comparison of genome-scale expression profile between wild-type and lhd2-1 plants provided a clue that LHD2 may regulate rice shoot development through KNOX and hormone-related genes. Further investigation will contribute to understand the action of LHD2 in controlling the shoot development of rice plants, which may also be conserved in grass species.

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Acknowledgements

We thank Jiayi Xie (Institute of Microbiology, Chinese Academy of Sciences) and Ying Lan (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for assistance in scanning electron microscopic observation. This work was supported by grants from National Natural Science Foundation of China (30330040 and 30221002).