Genetic control of compound leaf development in the mungbean (Vigna radiata L.)

Abstract

Many studies suggest that there are distinct regulatory processes controlling compound leaf development in different clades of legumes. Loss of function of the LEAFY (LFY) orthologs results in a reduction of leaf complexity to different degrees in inverted repeat-lacking clade (IRLC) and non-IRLC species. To further understand the role of LFY orthologs and the molecular mechanism in compound leaf development in non-IRLC plants, we studied leaf development in unifoliate leaf (un) mutant, a classical mutant of mungbean (Vigna radiata L.), which showed a complete conversion of compound leaves into simple leaves. Our analysis revealed that UN encoded the mungbean LFY ortholog (VrLFY) and played a significant role in leaf development. In situ RNA hybridization results showed that STM-like KNOXI genes were expressed in compound leaf primordia in mungbean. Furthermore, increased leaflet number in heptafoliate leaflets1 (hel1) mutants was demonstrated to depend on the function of VrLFY and KNOXI genes in mungbean. Our results suggested that HEL1 is a key factor coordinating distinct processes in the control of compound leaf development in mungbean and its related non-IRLC legumes.

Fig. 1. The ontogeny of compound leaf development in wild-type mungbean.

a Whole plant morphology of mungbean. The arrow indicates the opposite juvenile leaves at the first node. b A pair of juvenile leaves of mungbean. c Adult compound leaf of mungbean. d–i SEM analysis of compound leaf development. d Sites of the incipient leaf primordia were specified at the periphery of the SAM at S0. At S1, a common leaf primordium was initiated as a strip of cells outgrowing along the periphery of SAM. e A pair of stipule primordia (ST) was initiated from the proximal end of the common leaf primordium at S2. f At S3, the boundaries (arrows) between the stipule and lateral leaflet primordia were formed. g At S4, a pair of lateral leaflet primordia (LL) emerged between the stipule and common leaf primordium. h At S5, the common leaf primordium differentiated into a terminal leaflet primordium (TL) as indicated by development of trichomes from the abaxial surface. Boundaries (arrows) were formed between the lateral and terminal leaflet primordia. i At S6, the leaflet primordia folded as a result of outgrowth of the abaxial surface, and the region between the stipule and lateral leaflet primordia expanded to form a petiole (P). Trichomes developed from the abaxial surface of both the stipule and lateral leaflet primordia. b, c Scale bars = 1 cm; d–i Scale bars = 50 μm

Fig. 2. Phenotypes of mungbean unifoliate (un) mutants.

a Wild-type mungbean (left) and un1-1 mutant (right) exhibiting compound and simple leaf forms, respectively. Arrows indicate the opposite juvenile leaves at the first node in the wild type and at the first and second nodes in the un1-1 mutant. b Close-up views of the adult leaves of the wild type (left) and un1-1 mutant (right). c Morphology of a mature un1-1 mutant (right), exhibiting simple leaf and floral homeotic phenotypes compared to the wild type (left). The inset has a close-up view of the inflorescence of the un1-1 mutant. d, f Leaf development of wild type at S4 and S6. e, h Leaf development of un1-1 at S4 and S6. The lateral leaflet primordia did not form at the proximal end of the common leaf primordium. LL lateral leaflet, TL terminal leaflet, SL single leaflet. b–c Scale bars = 10 cm; d–f 50 μm indicated

Fig. 3. Molecular cloning of the VrLFY gene from mungbean.

a PCR amplification of the VrLFY gene from wild-type mungbean and un mutants (WT, un1-1, un1-2, un1-3, and un1-4). Deletions were detected as no product on attempted amplification of the VrLFY gene from three mutant alleles. b Phylogenetic analysis of VrLFY and its putative orthologs: VaLFY of V. angularis, PvLFY of Phaseolus vulgaris, GmLFY1 and GmLFY2 of soybean, PFM of L. japonicas, UNI of pea, SGL1 of M. truncatula, VFL of Vitis vinifera, FALSIFLORA of tomato, FLORICAULA of snapdragon, LEAFY of Arabidopsis, ChLFY of C. hirsute, RFL of rice and NEEDLY of Pinus radiata. Bootstrap supports above 50 % from 1000 replicates are shown. c, d VrLFY gene expression was detected in SAM and developing leaf primordia. e The VrLFY sense probes were used as a negative control, and no hybridization signal was detected in SAM and leaf primordia. Scale bars = 100 μm

Fig. 4. RNA in situ hybridization analysis of STM/BP-like KNOXI gene expression in mungbean.

RNA in situ hybridization analysis of a Vradi07g26830, b Vradi10g07810, c Vradi06g03570, and d Vradi06g14320 in the vegetative apices of mungbean. No expression was detected using the control sense probes of e Vradi07g26830, f Vradi10g07810, g Vradi06g03570, and h Vradi06g14320. Scale bars = 100 μm

Fig. 5. Genetic interactions among leaf mutants in mungbean.

Compound leaf phenotype of the wild type and mutants. a–f Mature compound leaves of a WT, b hel1-1, c smp1-1, d hel1-1 smp1-1, e un1-1, and f hel1-1 un1-1 mutants (all in the Sulu ecotype). Leaves of the hel1-1 un1-1 double mutants exhibited three leaflets with short petioles. The hel1-1 smp1-1 double mutants were heptafoliate leaves of small size, indicating an epistatic interaction between hel1 and smp1 in the control of leaflet number. Scale bars = 10 cm

Fig. 6. qRT-PCR analysis of key genes expressed in hel mutants.

qRT-PCR analysis of gene expression relative to that of the mungbean TUB gene. The level of transcripts was examined in the shoot apices of mutants compared with wild-type plants 2 weeks after seed germination. Bars represent means ± SEs (n = 3). Vradi07g26830 is a KNOXM gene. Vradi07g26830, Vradi10g07810, Vradi06g03570, and Vradi06g14320 are STM/BP-like KNOXI genes