Physiological and structural traits contribute to thermotolerance in wild Australian cotton species.

BACKGROUND AND AIMS: Five species of cotton (Gossypium) were exposed to 38°C days during early vegetative development. Commercial cotton (Gossypium hirsutum) was contrasted with four wild cotton species (G. australe, G. bickii, G. robinsonii and G. sturtianum) that are endemic to central and northern Australia. METHODS: Plants were grown at daytime maxima of 30°C or 38°C for 25 d, commencing at the four-leaf stage. Leaf areas and shoot biomass were used to calculate relative rates of growth and specific leaf areas. Leaf gas exchange measurements revealed assimilation and transpiration rates, as well as electron transport rates (ETR) and carboxylation efficiency (CE) in steady-state conditions. Finally, leaf morphological traits (mean leaf area and leaf shape were quantified), along with leaf surface decorations, imaged using scanning electron microscopy. KEY RESULTS: Shoot morphology was differentially affected by heat, with three of the four wild species growing faster at 38°C than at 30°C, whereas early growth in G. hirsutum was severely inhibited by heat. Areas of individual leaves and leaf numbers both contributed to these contrasting growth responses, with fewer, smaller leaves at 38°C in G. hirsutum. CO2 assimilation and transpiration rates of G. hirsutum were also dramatically reduced by heat. Cultivated cotton failed to achieve evaporative cooling, contrasting with the transpiration-driven cooling in the wild species. Heat substantially reduced ETR and CE in G. hirsutum, with much smaller effects in the wild species. We speculate that leaf shape, as assessed by invaginations of leaf margins, and leaf size contributed to heat dispersal differentially among the five species. Similarly, reflectance of light radiation was also highly distinctive for each species. CONCLUSIONS: These four wild Australian relatives of cotton have adapted to hot days that are inhibitory to commercial cotton, deploying a range of physiological and structural adaptations to achieve accelerated growth at 38°C.

Comparison between computer recognition and manual measurement methods for the estimation of leaf area.

BACKGROUND AND AIMS: Leaf area (A) is a crucial indicator of the photosynthetic capacity of plants. The Montgomery equation (ME), which hypothesizes that A is proportional to the product of leaf length (L) and width (W), is a valid tool for nondestructively measuring A for many broad-leaved plants. At present, the methods used to compute L and W for ME can be broadly divided into two kinds: using computer recognition, and measuring manually. However, the potential difference in the prediction accuracy using either method has not been thoroughly examined in prior studies. METHODS: In the present study, we measured 540 Alangium chinense leaves, 489 Liquidambar formosana leaves, and 215 Liriodendron × sinoamericanum leaves, utilizing computer recognition and manual measurement methods to determine L and W. ME was used to fit the data determined by the two methods, and the goodness of fits were compared. The prediction errors of A were analyzed by examining the correlations with two leaf symmetry indices (areal ratio of the left side to the right side, and standardized index for bilateral asymmetry), as well as the leaf shape complexity index (the leaf dissection index). KEY RESULTS: The results indicate that there is a neglectable difference in the estimation of A between both methods. This further validates that ME is an effective method for estimating A in broad-leaved tree species, including those with lobes. Additionally, leaf shape complexity significantly influenced the estimation of A. CONCLUSIONS: These results show that the use of computer recognition and manual measurement in the field are both effective and feasible, although the influence of leaf shape complexity should be considered when applying ME to estimate A in the future.

Genetic architecture of heritable leaf microbes.

Host-associated microbiomes are shaped by both their environment and host genetics, and often impact host performance. The scale of host genetic variation important to microbes is largely unknown yet fundamental to the community assembly of host-associated microbiomes, with implications for the eco-evolutionary dynamics of microbes and hosts. Using Ipomoea hederacea, ivyleaf morning glory, we generated matrilines differing in quantitative genetic variation and leaf shape, which is controlled by a single Mendelian locus. We then investigated the relative roles of Mendelian and quantitative genetic variation in structuring the leaf microbiome and how these two sources of genetic variation contributed to microbe heritability. We found that despite large effects of the environment, both Mendelian and quantitative genetic host variation contribute to microbe heritability and that the cumulative small effect genomic differences due to matriline explained as much or more microbial variation than a single large effect Mendelian locus. Furthermore, our results are the first to suggest that leaf shape itself contributes to variation in the abundances of some phyllosphere microbes.IMPORTANCEWe investigated how host genetic variation affects the assembly of Ipomoea hederacea's natural microbiome. We found that the genetic architecture of leaf-associated microbiomes involves both quantitative genetic variation and Mendelian traits, with similar contributions to microbe heritability. The existence of Mendelian and quantitative genetic variation for host-associated microbes means that plant evolution at the leaf shape locus or other quantitative genetic loci has the potential to shape microbial abundance and community composition.

Intra-leaf modeling of Cannabis leaflet shape produces leaf models that predict genetic and developmental identities.

The iconic, palmately compound leaves of Cannabis have attracted significant attention in the past. However, investigations into the genetic basis of leaf shape or its connections to phytochemical composition have yielded inconclusive results. This is partly due to prominent changes in leaflet number within a single plant during development, which has so far prevented the proper use of common morphometric techniques. Here, we present a new method that overcomes the challenge of nonhomologous landmarks in palmate, pinnate, and lobed leaves, using Cannabis as an example. We model corresponding pseudo-landmarks for each leaflet as angle-radius coordinates and model them as a function of leaflet to create continuous polynomial models, bypassing the problems associated with variable number of leaflets between leaves. We analyze 341 leaves from 24 individuals from nine Cannabis accessions. Using 3591 pseudo-landmarks in modeled leaves, we accurately predict accession identity, leaflet number, and relative node number. Intra-leaf modeling offers a rapid, cost-effective means of identifying Cannabis accessions, making it a valuable tool for future taxonomic studies, cultivar recognition, and possibly chemical content analysis and sex identification, in addition to permitting the morphometric analysis of leaves in any species with variable numbers of leaflets or lobes.

Shaping leaves through TALE homeodomain transcription factors.

The first TALE homeodomain transcription factor gene to be described in plants was maize knotted1 (kn1). Dominant mutations in kn1 disrupt leaf development, with abnormal knots of tissue forming in the leaf blade. kn1 was found to be expressed in the shoot meristem but not in a peripheral region that gives rise to leaves. Furthermore, KN1 and closely related proteins were excluded from initiating and developing leaves. These findings were a prelude to a large body of work wherein TALE homeodomain proteins have been identified as vital regulators of meristem homeostasis and organ development in plants. KN1 homologues are widely represented across land plant taxa. Thus, studying the regulation and mechanistic action of this gene class has allowed investigations into the evolution of diverse plant morphologies. This review will focus on the function of TALE homeodomain transcription factors in leaf development in eudicots. Here, we discuss how TALE homeodomain proteins contribute to a spectrum of leaf forms, from the simple leaves of Arabidopsis thaliana to the compound leaves of Cardamine hirsuta and species beyond the Brassicaceae.

Leaf morphological traits show greater responses to changes in climate than leaf physiological traits and gas exchange variables.

Adaptation to changing conditions is one of the strategies plants may use to survive in the face of climate change. We aimed to determine whether plants' leaf morphological and physiological traits/gas exchange variables have changed in response to recent, anthropogenic climate change. We grew seedlings from resurrected historic seeds from ex-situ seed banks and paired modern seeds in a common-garden experiment. Species pairs were collected from regions that had undergone differing levels of climate change using an emerging framework-Climate Contrast Resurrection Ecology, allowing us to hypothesise that regions with greater changes in climate (including temperature, precipitation, climate variability and climatic extremes) would be greater trait responses in leaf morphology and physiology over time. Our study found that in regions where there were greater changes in climate, there were greater changes in average leaf area, leaf margin complexity, leaf thickness and leaf intrinsic water use efficiency. Changes in leaf roundness, photosynthetic rate, stomatal density and the leaf economic strategy of our species were not correlated with changes in climate. Our results show that leaves do have the ability to respond to changes in climate, however, there are greater inherited responses in morphological leaf traits than in physiological traits/variables and greater responses to extreme measures of climate than gradual changes in climatic means. It is vital for accurate predictions of species' responses to impending climate change to ensure that future climate change ecology studies utilise knowledge about the difference in both leaf trait and gas exchange responses and the climate variables that they respond to.

PpSCARECROW1 (PpSCR1) regulates leaf blade and mid-vein development in Physcomitrium patens.

In plants, asymmetric cell divisions result in distinct cell fates forming large and small daughter cells, adding to the cellular diversity in an organ. SCARECROW (SCR), a GRAS domain-containing transcription factor controls asymmetric periclinal cell divisions in flowering plants by governing radial patterning of ground tissue in roots and cell proliferation in leaves. Though SCR homologs are present across land plant lineages, the current understanding of their role in cellular patterning and leaf development is mostly limited to flowering plants. Our phylogenetic analysis identified three SCR homologs in moss Physcomitrium patens, amongst which PpSCR1 showed highest expression in gametophores and its promoter activity was prominent at the mid-vein and the flanking leaf blade cells pointing towards its role in leaf development. Notably, out of the three SCR homologs, only the ppscr1 knock-out lines developed slender leaves with four times narrower leaf blade and three times thicker mid-vein. Detailed histology studies revealed that slender leaf phenotype is either due to the loss of anticlinal cell divisions or failure of periclinal division suppression in the leaf blade. RNA-Seq analyses revealed that genes responsible for cell division and differentiation are expressed differentially in the mutant. PpSCR1 overexpression lines exhibited significantly wider leaf lamina, further reconfirming the role in leaf development. Together, our data suggests that PpSCR1 is involved in the leaf blade and mid-vein development of moss and that its role in the regulation of cell division and proliferation is ancient and conserved among flowering plants and mosses.

Species identification through deep learning and geometrical morphology in oaks (Quercus spp.): Pros and cons.

Plant phenotypic characteristics, especially leaf morphology of leaves, are an important indicator for species identification. However, leaf shape can be extraordinarily complex in some species, such as oaks. The great variation in leaf morphology and difficulty of species identification in oaks have attracted the attention of scientists since Charles Darwin. Recent advances in discrimination technology have provided opportunities to understand leaf morphology variation in oaks. Here, we aimed to compare the accuracy and efficiency of species identification in two closely related deciduous oaks by geometric morphometric method (GMM) and deep learning using preliminary identification of simple sequence repeats (nSSRs) as a prior. A total of 538 Asian deciduous oak trees, 16 Q. aliena and 23 Q. dentata populations, were firstly assigned by nSSRs Bayesian clustering analysis to one of the two species or admixture and this grouping served as a priori identification of these trees. Then we analyzed the shapes of 2328 leaves from the 538 trees in terms of 13 characters (landmarks) by GMM. Finally, we trained and classified 2221 leaf-scanned images with Xception architecture using deep learning. The two species can be identified by GMM and deep learning using genetic analysis as a priori. Deep learning is the most cost-efficient method in terms of time-consuming, while GMM can confirm the admixture individuals' leaf shape. These various methods provide high classification accuracy, highlight the application in plant classification research, and are ready to be applied to other morphology analysis.

PLSR-colorimetric simultaneous determination of L-Tyrosine and L-Tryptophan in different pharmaceutical and biological samples using one-pot synthesized leaf shape Ag@Ag2O core-shell nanocomposites modified by ß-CD.

In the present study, a simple, innovative, and economically beneficial method has been proposed for the synthesis of Ag@Ag2O core-shell nanocomposites using Acanthophora muscoides algae extract. The host-guest recognition of targets was performed by modification of the Ag@Ag2O surface using ß-CD. The Ag@Ag2O- ß-CD NCs were used as a colorimetric sensor to determine L-Tryptophan and L-Tyrosine using a partial least square (PLS) approach. A crystalline hybrid structure of Ag core and an Ag2O shell was confirmed by XRD, FTIR, TEM and AFM research. Also, DLS analysis and surface zeta potential spectra illustrated the aggregated nature of nanocomposites in the presence of analytes. The literature review shows that the colorimetric simultaneous determination of L-Tryptophan (L-Try) and L-Tyrosine (L-Tyr) has not been reported. The Ag@Ag2O- ß-CD sensor exhibited outstanding sensing capability in a broad linear range of 2.0 -200 µM for both amino acids and low detection limit of 0.32 and 0.51 µM, for L-Try and L-Tyr, respectively. The good sensitivity and excellent selectivity regarding possible interfering species, originated from the synergistic effect of host-guest recognition in combination with colorimetric sensing. Additionally, determination of analytes in various pharmaceutical, supplement and urine samples, approved the practical applicability of the constructed sensor. The computed results confirmed that colorimetric sensing in conjunction with a PLS technique was appropriate for the precise and accurate simultaneous determination of target amino acids in complex mixtures with RMSEP less than 2.5% and recovery in the range of 103-108% with R.S.D. values less than 3%.

The Competing Endogenous RNAs Regulatory Genes Network Mediates Leaf Shape Variation and Main Effector Gene Function in Mulberry Plant (Morus alba).

Mulberry plants (Morus alba) have leaf shapes, ranging from unlobed to lobed, which are crucial for yield, growth, and adaptability, indicating their ability to adapt to their environment. Competing endogenous RNAs (ceRNAs) constitute a web of RNAs within the organism's transcriptional regulatory system, including protein-coding genes (mRNAs), microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and others. In this study, samples for ceRNA sequencing were categorized into two groups: whole leaves and lobed leaves, each group with three replicates. In addition, we isolated, cloned, and characterized the precursor miRNA (miR156x) from the leaves of M. alba. miR156x precursor had a length of 107 base pairs and a minimum folding free energy of 50.27 kcal/mol. We constructed a pCAMBIA-35S-GUS-miR156x dual overexpression vector and established a transient transformation system for mulberry. At an optimal transformation solution (OD600 = 0.7), the GUS gene showed a higher expression in the leaves of transiently transformed mulberry with miR156x overexpression, four days after transformation, while the target genes of miR156x had decreased expression in the same leaves. Investigations into the transgenic mulberry plants uncovered various modifications to physio-chemical parameters including POD, SOD, PRO, MDA, soluble proteins and sugars, and chlorophyl content. miRNAs in the plants were found to act as negative regulators of gene expression in response to changes in leaf shape regulation, which was confirmed in vitro using dual-luciferase reporter assays. Subsequently, we cloned Maspl3 in vitro and conducted GST-Pull down assays, obtaining multiple proteins that interacted with the Maspl3 gene. This indicates that the miR156x/Maspl3/MSTRG.25812.1 regulatory module contributes to the differences in mulberry leaf shape.

Overview of molecular mechanisms of plant leaf development: a systematic review.

Leaf growth initiates in the peripheral region of the meristem at the apex of the stem, eventually forming flat structures. Leaves are pivotal organs in plants, serving as the primary sites for photosynthesis, respiration, and transpiration. Their development is intricately governed by complex regulatory networks. Leaf development encompasses five processes: the leaf primordium initiation, the leaf polarity establishment, leaf size expansion, shaping of leaf, and leaf senescence. The leaf primordia starts from the side of the growth cone at the apex of the stem. Under the precise regulation of a series of genes, the leaf primordia establishes adaxial-abaxial axes, proximal-distal axes and medio-lateral axes polarity, guides the primordia cells to divide and differentiate in a specific direction, and finally develops into leaves of a certain shape and size. Leaf senescence is a kind of programmed cell death that occurs in plants, and as it is the last stage of leaf development. Each of these processes is meticulously coordinated through the intricate interplay among transcriptional regulatory factors, microRNAs, and plant hormones. This review is dedicated to examining the regulatory influences of major regulatory factors and plant hormones on these five developmental aspects of leaves.

Leaf shape, planting density, and nitrogen application affect soybean yield by changing direct and diffuse light distribution in the canopy.

When attempting to maximize the crop yield from field-grown soybean (Glycine max (L.) Merr.) by means of improving the light conditions for photosynthesis in the canopy, it is crucial to find the optimal planting density and nitrogen application rate. The soybean plants that were the subject of our experiment were cultivated in N-dense mutual pairs, and included two cultivars with different leaf shapes; one cultivar sported ovate leaves (O-type) and the other lanceolate leaves (L-type). We analyzed the results quantitatively to determine the amount of spatial variation in light distribution and photosynthetic efficiency across the canopy, and to gauge the effect of the experimental parameters on the yield as well as the photosynthetic light and nitrogen use efficiency of the crop. Results indicate that the different leaf shapes were responsible for significant disparities between the photosynthetic utilization of direct and diffuse light. As the nitrogen fertilizer rate and the planting density increased, the soybean plants responded by adjusting leaf morphology in order to maximize the canopy apparent photosynthetic light use efficiency, which in turn affected the leaf nitrogen distribution in the canopy. Despite the fact that the light interception rate of the canopy of the L-type cultivar was lower than that of the canopy of the O-type cultivar, we found its canopy apparent photosynthetic nitrogen and light use efficiency were higher. It was interesting to note, however, that the nitrogen and light use efficiency contributions associated with exposure to diffuse light were greater for the latter than for the former.

Brassinosteroid catabolic enzyme CYP734A129 regulates the morphologies of leaves and floral organs in woodland strawberry.

Brassinosteroids (BRs) play critical roles in plant growth and development and regulate many important agronomic traits. However, the functions of BRs in strawberry are unclear. This study identified two mutants, named P6 and R87, in woodland strawberry (Fragaria vesca) from EMS mutagenesis populations that exhibit narrow leaves, petals and sepals. Mapping by sequencing and genetic studies revealed that the F. vesca CYP734A129, encoding a putative BR catabolic enzyme, is the causative gene for both P6 and R87. Overexpression of CYP734A129 in both F. vesca and Arabidopsis causes a severe dwarf phenotype, and the BRI1-EMS-SUPPRESSOR 1 (BES1) protein is less abundant in the CYP734A129-overexpressing Arabidopsis seedlings. This suggests that CYP734A129 is functionally conserved with CYP734A1, as a BR-inactivating enzyme. Transcriptome analysis of young leaves revealed that four BR biosynthetic genes were significantly downregulated in P6 (cyp734a129), and photosynthesis-related genes were highly enriched among the up-regulated genes in P6 compared to the wild type. This further supports that CYP734A129 inactivates BRs in F. vesca. Furthermore, we showed that mutations in CYP734A129 do not affect fruit shape and color during ripening in strawberry. Overall, our results suggest that F. vesca CYP734A129 is a BR catabolic enzyme, and provide insights into the roles of CYP734A129 in strawberry.

Patterns of host plant use do not explain mushroom body expansion in Heliconiini butterflies.

The selective pressures leading to the elaboration of downstream, integrative processing centres, such as the mammalian neocortex or insect mushroom bodies, are often unclear. In Heliconius butterflies, the mushroom bodies are two to four times larger than those of their Heliconiini relatives, and the largest known in Lepidoptera. Heliconiini lay almost exclusively on Passiflora, which exhibit a remarkable diversity of leaf shape, and it has been suggested that the mushroom body expansion of Heliconius may have been driven by the cognitive demands of recognizing and learning leaf shapes of local host plants. We test this hypothesis using two complementary methods: (i) phylogenetic comparative analyses to test whether variation in mushroom body size is associated with the morphological diversity of host plants exploited across the Heliconiini; and (ii) shape-learning experiments using six Heliconiini species. We found that variation in the range of leaf morphologies used by Heliconiini was not associated with mushroom body volume. Similarly, we find interspecific differences in shape-learning ability, but Heliconius are not overall better shape learners than other Heliconiini. Together these results suggest that the visual recognition and learning of host plants was not a main factor driving the diversity of mushroom body size in this tribe.

Assessment of halophyte plant phenotypic responses under heavy metals pollution. Implications for monitoring and phytoremediation.

While phytoremediation is a highly valued practice to address local pollution problems, the use of early biomarkers of stress is useful for monitoring environments since they allow us to take measures before deleterious effects are irreversible. In this framework the goals are: to evaluate the pattern of leaf shape variation of Limonium brasiliense plants related to a metal soil gradient in the San Antonio salt marsh; to assess whether seeds from sites with different pollution levels show the same pattern of leaf shape variations under optimal growing conditions; and to compare the growth, the Pb accumulation pattern, and the leaf shape variation pattern of plants germinated from seeds originated in sites with different pollution levels in response to an experimental Pb rise. The results obtained from leaves collected in the field showed that the leaf shape changed depending on the soil metal levels. Plants germinated from seeds collected at the different sites expressed all the variation in leaf shape independently of the origin site, and the mean shape of each site was close to the consensus. Instead, when looking for the leaf shape components that maximize the differences between the sites from a growth experiment exposed to an increase in Pb in the irrigation solution, the pattern of variation found in the field disappeared. That is, only plants from the polluted site did not show variations in leaf shape in response to Pb additions. Finally, Pb accumulation in the roots was highest in plants germinated from seeds from the site where the soil pollution is greater. That suggests that seeds of L. brasiliense from polluted sites are better to use in phytoremediation practices, specifically to stabilize Pb in its roots whilst plants from the non-polluted site are better to detect pollutant soils using the leaf shape as an early biomarker.

The genetic basis of shoot architecture in soybean.

Shoot architecture refers to the three-dimensional body plan of the above ground organs of the plant. The patterning of this body plan results from the tight genetic control of the size and maintenance of meristems, the initiation of axillary growth, and the timing of developmental phase transition. Variation in shoot architecture can result in dramatic differences in plant productivity and/or grain yield due to their effects on light interception, photosynthetic efficiency, response to agronomic inputs, and environmental adaptation. The fine-tuning of shoot architecture has consequently been of great interest to plant breeders, driving the need for deeper understanding of the genes and molecular mechanisms governing these traits. In soybean, the world's most important oil and protein crop, major components of shoot architecture include stem growth habit, plant height, branch angle, branch number, leaf petiole angle, and the size and shape of leaves. Key genes underlying some of these traits have been identified to integrate hormonal, developmental, and environmental signals modulating the growth and orientation of shoot organs. Here we summarize the current knowledge and recent advances in the understanding of the genetic control of these important architectural traits in soybean.

Multiple Leaf Sample Extraction System (MuLES): A tool to improve automated morphometric leaf studies.

Premise: The measurement of leaf morphometric parameters from digital images can be time-consuming or restrictive when using digital image analysis softwares. The Multiple Leaf Sample Extraction System (MuLES) is a new tool that enables high-throughput leaf shape analysis with minimal user input or prerequisites, such as coding knowledge or image modification. Methods and Results: MuLES uses contrasting pixel color values to distinguish between leaf objects and their background area, eliminating the need for color threshold-based methods or color correction cards typically required in other software methods. The leaf morphometric parameters measured by this software, especially leaf aspect ratio, were able to distinguish between large populations of different accessions for the same species in a high-throughput manner. Conclusions: MuLES provides a simple method for the rapid measurement of leaf morphometric parameters in large plant populations from digital images and demonstrates the ability of leaf aspect ratio to distinguish between closely related plant types.

Anatomical determinants of gas exchange and hydraulics vary with leaf shape in soybean.

BACKGROUND AND AIMS: Leaf shape in crops can impact light distribution and carbon capture at the whole plant and canopy level. Given similar leaf inclination, narrow leaves can allow a greater fraction of incident light to pass through to lower canopy leaves by reducing leaf area index, which can potentially increase canopy-scale photosynthesis. Soybean has natural variation in leaf shape which can be utilized to optimize canopy architecture. However, the anatomical and physiological differences underlying variation in leaf shape remain largely unexplored. METHODS: In this study, we selected 28 diverse soybean lines with leaf length to width ratios (leaf ratio) ranging between 1.1 and 3.2. We made leaf cross-sectional, gas exchange, vein density and hydraulic measurements and studied their interrelationships among these lines. KEY RESULTS: Our study shows that narrow leaves tend to be thicker, with an ~30 µm increase in leaf thickness for every unit increase in leaf ratio. Interestingly, thicker leaves had a greater proportion of spongy mesophyll while the proportions of palisade and paraveinal mesophyll decreased. In addition, narrow and thicker leaves had greater photosynthesis and stomatal conductance per unit area along with greater leaf hydraulic conductance. CONCLUSIONS: Our results suggest that selecting for narrow leaves can improve photosynthetic performance and potentially provide a yield advantage in soybean.

Insight into the formation of trumpet and needle-type leaf in Ginkgo biloba L. mutant.

The leaf type of a plant determines its photosynthetic efficiency and adaptation to the environment. The normal leaves of modern Ginkgo biloba, which is known as a "living fossil" in gymnosperm, evolved from needle-like to fan-shaped with obvious dichotomous venation. However, a newly discovered Ginkgo variety "SongZhen" have different leaf types on a tree, including needle-, trumpet-, strip-, and deeply split fan-shaped leaves. In order to explore the mechanism in forming these leaf types, the microscopy of different leaf types and transcriptome analysis of apical buds of branches with normal or abnormal leaves were performed. We found that the normal leaf was in an intact and unfolded fan shape, and the abnormal leaf was basically split into two parts from the petiole, and each exhibited different extent of variation. The needle-type leaves were the extreme, having no obvious palisade and spongy tissues, and the phloem cells were scattered and surrounded by xylem cells, while the trumpet-type leaves with normal vascular bundles curled inward to form a loop from the abaxial to adaxial side. The other type of leaves had the characteristics among needle-type, trumpet-type, or normal leaves. The transcriptome analysis and quantitative PCR showed that the genes related to abaxial domain were highly expressed, while the adaxial domain promoting genes were decreasingly expressed in abnormal-type leaf (ANL) buds and abnormal leaves, which might lead to the obvious abaxialized leaves of "SongZhen." In addition, the low expression of genes related to leaf boundary development in ANL buds indicated that single- or double-needle (trumpet) leaves might also be due to the leaf tissue fusion. This study provides an insight into the mechanism of the development of the abnormal leaves in "SongZhen" and lays a foundation for investigating the molecular mechanism of the leaf development in gymnosperms.