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Function of the 'double' leaf on a pomelo tree

Function of the 'double' leaf on a pomelo tree



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What is the function of the 'double leaf' (I'm unsure what the technical name is… ) on a pomelo tree leaf?

Furthermore, what evolutionary 'problem' is this structure solving?

See image with arrows that indicate the two parts to the leaf.


The functional difference of eight chitinase genes between male and female of the cotton mealybug, Phenacoccus solenopsis

Correspondence: Dr Fei Li, Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects/Institute of Insect Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China. e-mail: [email protected]

Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects/Institute of Insect Science, Zhejiang University, Hangzhou, China

Department of Plant Protection, Faculty of Agriculture (Saba Basha), Alexandria University, Alexandria, Egypt

Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects/Institute of Insect Science, Zhejiang University, Hangzhou, China

Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects/Institute of Insect Science, Zhejiang University, Hangzhou, China

Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects/Institute of Insect Science, Zhejiang University, Hangzhou, China

Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects/Institute of Insect Science, Zhejiang University, Hangzhou, China

Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects/Institute of Insect Science, Zhejiang University, Hangzhou, China

Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects/Institute of Insect Science, Zhejiang University, Hangzhou, China

Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects/Institute of Insect Science, Zhejiang University, Hangzhou, China

Correspondence: Dr Fei Li, Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects/Institute of Insect Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China. e-mail: [email protected]

Abstract

The cotton mealybug Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) is a polyphagous insect that attacks tens of plant and causes substantial economic loss. Insect chitinases are required to remove the old cuticle to allow for continued growth and development. Though insect chitinases have been well studied in tens of insects, their functions in mealybug are still not addressed. Here, we sequenced the transcriptomes of adult males and females, from which eight chitinase genes were identified. We then used the method of rapid amplification of cDNA ends to amplify their full length. Phylogenetic analysis indicated that these genes clustered into five subgroups. Among which, group II PsCht2 had the longest transcript and was highly expressed at second instar nymph. PsCht10, PsCht3-3 and PsIDGF were highly expressed in the adult females, whereas PsCht4 and PsCht4-1 were significantly expressed at the male pupa and adult male. Next, we knocked down all eight chitinase genes by feeding the double-stranded RNA. Knockdown of PsCht4 or PsCht4-1 led to the failure of moult and, silencing PsCht5 resulted in pupation defect, while silencing PsCht10 led to small body size, suggesting these genes have essential roles in development and can be used as a potential target for pest control.

Fig. S1 The domain architecture of chitinase genes from P. solenopsis. The chitinases identified have the conserved domain of glycoside hydrolase family 18, and the domain structure analysis was carried out by searching the Pfam database and then checked with HMMER and SMART.

Fig. S2 The experimental device for dsRNA feeding. After washing with distilled water, cotton leaves were immersed at the petiole end in 1.5 ml Eppendorf tube containing 400 μl RNase-free water mixed with either dsRNA or dsGFP (about 0.1μg/μl). Thirty third instar nymph were allowed to resume feeding on these green leaves.

Table S1 Transcriptome data accession number

Table S2 Homologs of sequences for phylogenetic trees

Table S3 Counts number of the chitinase genes in cotton mealybug among adult male and adult female

Table S4 Similarities of RNAi-lethal genes with the orthologous in other insects

Table S5 Entire dsRNA sequences and primers that used for RNAi experiment

Table S6 Entire dsRNA sequences and primers that used for RNAi experiment

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


II. Overall structure of the leaf venation

Veins are composed of xylem and phloem cells embedded in parenchyma, sometimes sclerenchyma, and surrounded by bundle sheath cells. The vein xylem transports water from the petiole throughout the lamina mesophyll, and the phloem transports sugars out of the leaf to the rest of the plant. Leaf venation systems vary strongly across major plant lineages, with many early groups having dichotomously branching, open systems, but reticulation evolved frequently. Angiosperms have greatest diversity in vein structure but share key architectural elements, that is, a hierarchy of vein orders forming a reticulate mesh (Hickey, 1973 Ellis et al., 2009 McKown et al., 2010 ). Typically there are three orders of lower-order veins, known as ‘major veins’, often ribbed with sclerenchyma (Esau, 1977 ). One or more first-order veins run from the petiole to the leaf apex, with second-order veins branching at intervals, and third-order veins branching between. The major veins can be distinguished from minor veins, typically present only in angiosperms, which include up to four additional orders of smaller, reticulate higher-order veins. Major and minor veins can be distinguished by their distinct timing of formation, and differences in gene expression during development, sizes and branching in the mature leaf, and in cross-sectional anatomy (Esau, 1977 Haritatos et al., 2000a ). In several lineages, particularly the monocotyledons, a grid-like ‘striate’ venation is typical, including several orders of longitudinal veins of different sizes, with small transverse veins connecting them (Ueno et al., 2006 ).


Zippy Xylem

The xylem of a plant is the system of tubes and transport cells that circulates water and dissolved minerals. As a plant, you have roots to help you absorb water. If your leaves need water and they are 100 feet above the ground, it is time to put the xylem into action! Xylem is made of vessels that are connected end to end for the maximum speed to move water around. They also have a secondary function of support. When someone cuts an old tree down, they reveal a set of rings. Those rings are the remains of old xylem tissue, one ring for every year the tree was alive.


Methods

Simulation framework (microbiome data)

We simulate samples for two groups: control (C) and treatment (T), and generate OTU counts ( ( mathbf _^) or (mathbf _^) ) in a sample j from a Dirichlet-multinomial (DM) distribution with parameters estimated from a real microbial dataset, as has been suggested in several articles [16, 17]. The real throat data, throat_v35, is subset from V35 that is provided in the R package HMP16SData [37], by taking 153 samples collected from throat and 956 OTUs (operational taxonomic units) with non-zero count in more than 25% of samples. In particular, we sample:

where (mathbf _^ =left (x^_<1j>, dots, x^_<> ight)) and (mathbf _^ =left (x^_<1j>, dots, x^_<> ight)) are counts of K=956 OTUs in a sample j that belongs to control or treatment group, respectively nj is the total count of sample j that is randomly sampled from sequencing depths of 153 samples in throat_v35 (mathbf ^ =left (alpha ^_<1>, dots, alpha ^_ ight)) and (mathbf ^ =left (alpha ^_<1>, dots, alpha ^_ ight)) are parameters storing information about the relative abundance (proportion) and dispersion of OTUs in the control and treatment group, respectively. We estimate α C using the R package dirmult [38] that reparameterizes α C with (mathbf > = left (pi ^_<1>. pi ^_ ight)) and θ, where (pi ^_) is the expected proportion of OTU k in a sample belonging to the control group, and θ is a parameter about OTU correlation. In short, (alpha ^_ = pi ^_ frac <(1- heta)>< heta >) . In our simulation, θ is estimated from throat_v35 to apply in both control and treatment groups, and π C and π T are manipulated to create three scenarios: BS, US, and SS (see Fig. 2a and Additional file 1: Fig. S8). The simulated data (in the control group) is shown to have similar mean-variance relationship but a bit less random zeros when compared to the real data using countSimQC [39] (see Additional file 2).

In BS, signals are simulated on two randomly selected branches (A and B) by swapping their proportions in the treatment group as Eq. 2 US and SS are in Additional file 1: Supplementary Note 1.

where (r = frac hat ^_> hat ^_>) is the fold change (hat ^_) is the estimated proportion of OTU k from throat_v35. In other words, π C is estimated from throat_v35, and π T is obtained based on π C by changing values of selected OTUs.

Description of treeclimbR methodology

Data aggregation

Here, the aggregation is shown in Eqs. 3 and 4 for the DA and DS case, respectively. Depending on the dataset and method used in the differential analysis, the mean or median might be used instead of sum. In the DA case, counts of K entities in J samples are observed, and a tree on entities is constructed such that each entity can be mapped to a leaf. Data is aggregated in a way that the count of node i in sample j, Yij is generated as:

where b(i) represents the descendant leaves of node i (see tree notations in Fig. 1) M is the total number of nodes on the tree J is the number of samples K is the number of entities observed.

In the DS case, we have values of G features observed on each cell from J samples, and a tree about cell subpopulations (entities) is constructed such that multiple cells are mapped to a leaf. Samples are collected from different experiment conditions. The value of feature g on node (cell subpopulation) i in sample j, (Y^_<>) is aggregated from cells as:

where k ∈ (ji) means that a cell k is from sample j and belongs to subpopulation i (cell k is mapped to the descendant leaves of node i, or kb(i)) M, J, and K correspond to the total number of nodes, samples, and cells, respectively.

Differential analysis

Differential analysis is performed at all nodes of the tree. For the parametric synthetic microbial data and AML-sim data, we use edgeR to model the count data with negative binomial distribution and obtain P values via likelihood ratio tests for the following methods: BH, HFDR, minP, StructFDR, diffcyt, and treeclimbR. miLineage has its own way to calculate P values. For non-parametric synthetic microbial datasets, the non-parametric Wilcoxon rank sum test is used to compare the taxa’s abundance between two groups, which generates P values for all benchmarked methods. For BCR-XL-sim, the median transformed expressions of cell state markers on each node (cell subpopulation) of the tree are compared between groups using limma [40], which generates P values for diffcyt, minP, and treeclimbR. Three real datasets (Infant gut microbiota, mouse miRNA, and mouse cortex scRNA) are all count data, and edgeR is used for the differential analysis.

The generation of candidates

Candidates are used to capture the latent signal pattern on the tree. The search for candidates is based on a U score defined as Eq. 5:

Here, qk(t) is a score of node k, derived from its P value pk and estimated direction sign(θk), under a tuning parameter t. When pkt,qk(t)=1 with sign(θk) otherwise, qk(t)=0. The U score of node i at t, Ui(t), is the absolute average q scores over nodes in B(i) that includes node i and its descendant nodes. nB is the number of nodes in B(i). The U score could be considered as a measure of coordinate change within a branch. It achieves 1 when a consistent pattern, which includes both signs in the same direction and P values below t, is observed, and it is close to 0 when nodes in a branch highly disagree on either the sign or P value. With a suitable t value, we might expect signal branches are in a consistent pattern while others that have P values following a uniform distribution [0,1] and directions arbitrary up or down on leaves are not. Since signal branches are unknown in reality, we cannot directly determine the value of t. To suggest different candidates of signal branches, the tree is explored by tuning t in the range [0,1] (see Additional file 1: Fig. S17).

A candidate at t is obtained using the procedure below:

It starts from the root and moves toward leaves along edges.

For each path, it stops when a node i having Ui(t)=1 and pi<0.05 appears or the leaf is reached.

If a branch without signal by chance has the same direction, its branch node might reach U=1 at high t (e.g., t=1). In branches without signals, to keep candidate close to the leaf level, we hinder the selection of an internal node with a restriction pi<0.05. This means the probability of representing a three-leaf branch, without signals, using an internal node is around 0.01, and is much lower for a larger branch. P values selected in such a procedure are unbiased at different t for branches without signal and follow a uniform distribution (see Additional file 1: Fig. S16).

If multiple features exist, the procedure is carried out separately for each feature, and the global candidate at t, C(t), is defined as:

where Cg(t) is the candidate of feature g generated at t, and G includes all features.

The selection of candidates

Correction for multiple testing is performed separately on each candidate, but FDR is controlled on the leaf level by limiting t in the range as below (see Additional file 1: Supplementary Note 2 and Fig. S15).

where α is the nominal FDR r is the average size of signal branches identified at FDR=α. The branch size is the number of leaves in a branch. If r=1, signals do not cluster on the tree, and the leaf level (t=0) should be used. In real data, r is unknown and is estimated for a candidate C(t) as:

where s is the number of nodes with H0 rejected on the candidate C(t), and l is the number of descendant leaves of those rejected nodes.

Candidates that are generated with (t otin [0, 2 alpha (hat <>>-1)]) are firstly discarded to control FDR. Those that have reported the highest number of leaves with the lowest number of nodes are then selected to increase power while keeping results as short as possible.

The preprocessing and analysis of datasets

Available methods

For LEfSe, the default settings of LEfSe that is installed with conda in python 2.7 are used. For miLineage, we have applied both one-part (miLineage1) and two-part analysis (miLineage2) using the R package miLineagev2.1. For lasso, we build lasso-regularized logistic regression models, which consider values of features (e.g., abundance or expression) on all nodes of the tree as the explanatory variables, and the sample information (e.g, control or treatment group) as the response variable, with R package glmnet2.0-18 and chose model that gives the minimum mean cross-validated error. For diffcyt (v1.6.0), we use diffcyt’s testDA_edgeR and testDS_limma to analyze AML-sim and BCR-XL-sim datasets, respectively. For StructFDR and HFDR, R packages StructFDRv1.3 and structSSIv1.1.1 are used, respectively. Inputs on nodes (e.g., P values) required by methods StructFDR, HFDR, treeclimbR, and minP (see Additional file 1: Supplementary Note 3) are estimated by edgeRv3.28.0 (treeclimbR’s runDA function) in all datasets, except that diffcyt’s testDS_limma was used in BCR-XL-sim datasets. Unless specified, the default settings provided in R packages are used for all methods.

Parametric synthetic microbial data

To evaluate performance of methods on different signal patterns, datasets are simulated under three scenarios (BS, US, and SS) on two randomly selected branches using the R package treeclimbR’s simData function. More simulations with varying signal branches are provided to introduce signals on branches with different characteristics (see Additional file 1: Supplementary Note 1). Due to the swap of relative abundances between branches, the absolute logFC in BS, SS, and US are 1.45, 2.26, and in the range [0.02,2.13], respectively. For each scenario, 100 repetitions that are on the same signal branches but different counts on OTUs are made. To perform DA analysis, data was aggregated using Eq. 3.

AML-sim and BCR-XL-sim

Datasets were downloaded from the HDCytoData [41] R package. According to cell type markers, cells were first grouped into a large number of clusters (400,900,1600 in AML-sim datasets and 100, 400, 900 in BCR-XL-sim datasets) using FlowSOM [42]. Then, among clusters, pairwise euclidean distances were computed using their median expressions of type markers to generate a dissimilarity matrix. Finally, the hierarchical clustering from stats’s hclust [43] was applied on the matrix to create a tree on clusters.

Infant gut microbiota data

The data was downloaded from the curatedMetagenomicData [44] package that provides uniformly processed human microbiome data. Only samples from babies were used. This includes a count matrix with 464 metaOTUs in rows and 285 samples in columns, and a phylogenetic tree that has 464 leaves (metaOTUs) and 463 internal nodes. Samples belong to four time points: 4 days (0M), 4 months (4M), and 12 month (12M). At each time point, there are 15 samples from the C-section group and about 80 samples (80 in 0M, 81 in 4M, and 79 in 12M) from the vaginal group. Data was aggregated according to Eq. 3.

Mouse miRNA data

The data is from Kokkonen-Simon et al. [30], and 10 samples, including 5 receiving TOC and 5 receiving Sham surgery, are used. The trimming, alignment, and quantification of miRNA sequences were processed using sports [45], which ended up with 6375 miRNA sequences with counts in more than one sample. The tree was constructed based on the origins of the miRNA sequences: the miRNAs were grouped by primary transcript using the miRBase v22.1 annotation, and primary transcripts less than 10kb apart were further grouped into genomic clusters. It has 774 internal nodes and 6375 leaves. A leaf represents a unique sequence, and an internal node represents multiple sequences that share the same biological origin on a specific level. Data was aggregated as Eq. 3, and edgeR [46] was used to compare abundance between mice receiving TOC and mice receiving Sham surgery.

Mouse cortex scRNAseq data

We followed the preprocessing done by Crowell et al. [8] that annotates cells with 8 cell types. To obtain cell type markers, expressions of genes among cell types were first compared using FindAllMarkers (from Seuratv3.1.1) separately in each vehicle-treated sample to avoid selecting LPS-related state genes. For each cell type, the top 20 genes (ranked by absolute logFC) with absolute logFC above 0.5 were then selected We further removed markers that were only identified in one sample and finally obtained 125 marker genes. Based on 135 unique marker genes (13 canonical type marker genes and 125 computationally identified marker genes), a tree that encodes information of cell subpopulations at different resolutions was constructed using Seurat’sFindClusters (resolution at 6) and BuildClusterTree. The tree has 66 leaves, each of them representing a cell subpopulation. To perform DS analysis, data was aggregated as Eq. 4.


Abstract

L-PEACH is an L-system-based functional–structural model for simulating architectural growth and carbohydrate partitioning among individual organs in peach (Prunus persica (L.) Batsch) trees. The original model provided a prototype for how tree architecture and carbon economy could be integrated, but did not simulate peach tree architecture realistically. Moreover, evaluation of the functional characteristics of the individual organs and the whole tree remained a largely open issue. In the present study, we incorporated Markovian models into L-PEACH to improve the architecture of the simulated trees. The model was also calibrated to grams of carbohydrate, and tools for systematically displaying quantitative outputs and evaluating the behaviour of the model were developed. The use of the Markovian model concept to model tree architecture in L-PEACH reproduced tree behaviour and responses to management practices visually similar to trees in commercial orchards. The new architectural model along with several improvements in the carbohydrate-partitioning algorithms derived from the model evaluation significantly improved the results related to carbon allocation, such as organ growth, carbohydrate assimilation, reserve dynamics and maintenance respiration. The model results are now consistent within the modelled tree structure and are in general agreement with observations of peach trees growing under field conditions.

Additional keywords: architectural modelling, carbon allocation, carbon-based model, functional–structural plant modelling, peach tree growth simulation.


Cycas: Distribution, Morphology and Reproduction| Cycadales

In this article we will discuss about Cycas. After reading this article you will learn about: 1. Distribution of Cycas 2. General Morphology of Cycas 3.Anatomy of Vegetative Parts 4. Reproduction 5. Economic Importance.

  1. Distribution of Cycas
  2. General Morphology of Cycas
  3. Anatomy of Vegetative Parts of Cycas
  4. Reproduction of Cycas
  5. Economic Importance of Cycas

1. Distribution of Cycas:

Cycas, the largest genus among the Old World Cycads, is the most widely distributed genus of order Cycadales. It is distributed in Japan, Australia, India, Indochina, China, Mauritius, Africa, Nepal, Bangladesh, Sri Lanka and Myanmar. In India, Cycas grows naturally in Orissa, Assam, Meghalaya, Tamil Nadu, Karnataka and Andaman and Nicobar Islands (Fig. 8.7).

Cycas is represented by 15 species but according to Willis (1966) there are 20 species of the genus. Schuster (1932), however, recognizes only 8 species, mentioning for the rest as the forms, varieties or sub-species of the other species.

Besides Cycas circinalis, C pectinata, C. rumphii and C. beddomei, which occur in the wild state in India, C. revoluta and C. siamensis are such species which are cultivated commonly in the Indian gardens. Cycas revoluta is the most commonly cultivated species of the Indian gardens.

Some Indian Species:

1. Cycas beddomei Dyre:

A small shrub with a trunk of about 40 cm long. It is distributed in Andhra Pradesh, Madras, Calicut, etc. Leaves are large and reach up to 1 metre in length with quadrangular rachis. Leaflets are narrow and linear. Male cones are oblong to ovoid, bearing a short peduncle. Megasporophylls are ovate, lanceolate with dentate margins. They are produced in November-December.

2. Cycas circinalis Linn:

Commonly called ‘Jangli-madan-mast-ka-Phul’ (Hindi) or ‘Kamakshi’ (Telugu), C. circinalis is commonly distributed in western part of Peninsular India, Western Ghat and Orissa Hills in India. It is often cultivated in Indian gardens.

It is an evergreen tree bearing leaves of 1.5 to 3 metres in length with about 100 pairs of leaflets. Leaflets are linear-lanceolate with flat margin and acuminate apex. Upper sterile part of megasporophyll is longer than broad with dentate margins. Male cones are cylindrical to ovoid with a short peduncle. Megasporophylls contain brown tomentose hairs.

3. Cycas pectinata Griff:

It is distributed in Sikkim, Assam, Manipur and Someshwar Hills of Bihar in India along with some other countries including Nepal and Bangladesh. Its trunk ranges from 1.5 to 2.5 metres in length.

Leaves attain a length of about 1.5 to 2 metres. Leaflets are narrow, linear, tapering into a minute spine and measure from 14 to 25 cm. in length. Male cone is cylindrical-ovoid. The upper part of the megasporophyll is as broad as long.

4. Cycas revoluta Thunb:

It grows in wild state in Japan, China and Taiwan and is widely cultivated in several parts of the world, including India. It is so named because of the revoluted margins of its leaflets It is a palm-like tree, the trunk of which reaches up to 2 metres in length. Male cones are cylindrical or ovoid-oblong. Megasporophylls are 10-25 cm in length and densely tomentose

5. Cycas Rumphii Miq:

It is an evergreen palm-like tree distributed in Andaman and Nicobar Islands of India along with Sri Lanka, Malaysia and Australia. Its trunk reaches up to 4 metres while the leaves attain a length of 1-2 metres with 50 to 100 or more pairs of leaflets. Male cone is shortly stalked and ellipsoidal to oblong in shape. Megasporophylls are ovate-lanceolate with many small teeth.

6. Cycas siamensis Miq:

It is found in Myanmar, Thailand, China and Laos. It is a palm­ like tree. The leaves reach about 1 metre in length. Leaflets are narrow, linear with mucronate or acuminate apex. Male cone is ovoid oblong.

Megasporophyll’s sterile blade is as broad as long with usually only 2 ovules. Burkill (1933) considered Cycas siamensis as a geographical form of C. pectinata. Pant and Nautial (1963) also consider the two species similar, mainly because of their epidermal and anatomical studies.

2. General Morphology of Cycas:

Cycas is a palm-like, evergreen plant (Fig. 8.8). Prior to the anatomical studies of the stem of Cycas revoluta by Brongniart (1829), the Cycas was actually considered a palm. The plant body consists of a columnar aerial trunk with a crown of pinnately compound leaves as its top.

According of Eichler (1889), Coulter and Chamberlain (1910), Schuster (1932) and others, a tap root system persists in the adult plant, but according to Worsdell (1906) the tap roots are soon replaced by adventitious roots.

Roots in Cycas are of two types, i.e., normal tap roots forming a tap root system, and coralloid roots. Normal tap-roots are positively geotropic, grow deep into the soil and generally possess no root hairs. Their function is to fix the plant in the soil and to absorb water and other minerals.

From the normal roots develop some lateral branches near the ground surface. These lateral roots get infected with some bacteria, fungi and algae, and are called coralloid roots (Fig. 8.9). They grow- first horizontally in the soil and become swollen at their tips.

They divide repeatedly to form big bunches of greenish or brownish structures, which are coral like in appearance. They divide dichotomously, come out of the soil on the ground surface and are phototrophic in nature. Young plants bear more coralloid roots than the older ones.

Recently, Pant and Das (1990) reported non-coralloid aerial roots in Cycas circinalis, C. revoluta and C. rumphii. The charactenstic algal zone of coralloid roots is absent in these roots. These are positively geotropic, adventitious and develop from the lower sides of leaf bases or bulbils when they are still attached to the plant.

The stem is thick, woody and usually un-branched. It is tuberous when young but columnar, erect and stout at maturity. Branching in stem (Fig. 8.10) is also not rare after the plants have reached a certain age. The aerial part of the trunk remains covered by a thick armour of large and small rhomboidai leaf bases.

These occur regularly in alternate bands (Fig. 8 .11). The larger leaf bases represent the bases of foliage leaves, while the smaller ones are the bases of scaly leaves in male plants and scales and megasporophylls in female plants. The age of the plant can be calculated by counting the number of crowns of leaves and megasporophylls which are produced every year.

Among all Cycas species, C. media is tallest, attaining a height up to 20 metres. Regarding the age of Cycas, the plants can survive for a long period. C. circinalis, if allowed to grow undisturbed, may attain an age of 100 years or even more.

Two types of leaves are present in Cycas. These are green, assimilatory ox foliage leaves, and scaly leaves or cataphylls.

1. Foliage Leaves or Assimilatory Fronds:

These are green, large, pinnately compound and stout leaves with a spiny petiole and large, strong rachis. They are produced at the apex of the stem in the form of crown. The rachis bears many leaflets.

With the help of a transversely expanded rhomboidai leaf base, a leaf remains attached with the stem Two rows of strong and stiff spines are present on the petiole. These spines gradually transform into two rows of pinnae towards the upper side of the leaf (Fig. 8.12).

Pant (1953) reported many abnormalities in Cycas leaves. Author, along with two of his colleagues, also reported many abnormalities in the vegetative parts of an year-old plant of Cycas circinalis growing in the Botanical Garden of Meerut College, Meerut.

Cycas leaf is very large and may reach up to 3 metres in length in some species such as C. thouarsii. Two rows of pinnae on the leaves may be alternate or opposite. The number of pinnae varies in different species. As many as hundred pairs of pinnae may be present in a mature leaf.

Each pinna is sessile, elongated, ovate or lanceolate in shape with a spiny or acute apex. Pinnae are repeatedly and deeply dichotomized in C. micholitzii (Fig. 8.13). Each pinna or of leaflet contains a midrib without any lateral branching.

Forking of the midvein of the leaflet has been reported in C. circinalis by author in 1976. Margins of the leaflets are revolute in C. revoluta and C. beddomei, while in C. rumphii and C. circinalis they are flat.

According to Chamberlain (1935) the “vernation is circinate in the midrib and pinnules of Cycas”. Leaves, when young, have circinately coiled pinnae like those of ferns (Fig. 8.14). Very young parts of Cycas are also covered by fern-like hairs or ramenta.

2. Scaly Leaves or Cataphylls:

These are dry, brown-coloured, somewhat triangular leaves with their one end pointed. They are present at the apex of the stem and remain covered with several ramental hairs (Fig. 8.15).

3. Anatomy of Vegetative Parts of Cycas:

(i) Normal Root (Young):

It is circular in outline and resembles structurally with dicotyledons (Fig. 8.16). Outermost layer is epiblema or exodermis, which surrounds the large parenchymatous cortex. Epiblema consists of tangentially elongated cells. From some of its cells arise root hairs.

In the wide zone of parenchymatous cortex there are present many intercellular spaces. Cells of the cortex remain filled with starch. Some tannin-filled cells, mucilage cells and sometimes sphaeraphides are also present in the cortex. The cortex is delimited by a single- layered endodermis. Casparian steps are present in the barrel-shaped cells of the endodermis.

Endodermis is followed by multilayered pericycle. Xylem and phloem bundles in the roots are radially arranged, i.e. present on different radii. The roots are usually diarch but sometimes the number of protoxylem strands range between 3 to 8.

The protoxylem consists of spiral tracheids while the metaxylem consists of scalariform tracheids. Vessels are absent. Phloem is present alternately with xylem groups and consists of sieve tubes and phloem parenchyma. Pith is generally absent.

(ii) Normal Root (old) Showing Secondary Growth:

The older roots (Fig. 8.17) undergo secondary growth. The cambium cuts secondary phloem towards the outer side and secondary xylem towards the inner side. After sometime the pericycle cells also become meristematic and form a complete cambial ring.

The secondary xylem consists of radial rows of tracheids separated by parenchymatous cells. The crushed primary phloem is present in the form of dark streaks outside the secondary phloem. The secondary xylem is manoxvlic and contains many multiseriate rays.

Periderm starts to develop in the cortex of old roots. Some of the cells of the outermost cortical region start to become meristematic and function as cork cambium. It cuts cork towards outer side and secondary cortex towards inner side. Cork cells are dead and remain filled with subenn. Cycas roots often show two layers of periderm (Fig. 8.17).

Epiblema is ruptured and there are no root hairs in the older roots.

(iii) Coralloid Root:

Anatomically, the coralloid roots (Fig. 8.18) resemble normal roots except some under mentioned differences:

1. The secondary vascular tissue in coralloid roots is either totally absent or poorly-developed.

2. The cortex is wider in comparison with the normal root.

3. Presence of a greenish algal-zone in the middle of the cortex. But according to Chaudhary and Akhtar (1931) the algal-zone is not of universal occurrence in the coralloid roots of Cycas. It may be absent in such coralloid roots which go very deep in the soil. According to these workers only those coralloid roots are negatively geotropic which are infected by algal members.

Algal-zone consists of radially elongated, large, thin-walled cells having large intercellular spaces occupied by algae. Life (1901) opined that these spaces are formed because of the retardation of growth of such cells which are already infected by fungi and bacteria.

Such infected cells cannot keep pace with the neighbouring cells, and a tension is produced which results in the formation of air spaces by breaking of certain cells. These spaces are further widened by the algal infection. But according to Chaudhary and Akhtar (1931) the alga is mainly responsible for the formation of these large intercellular spaces.

Following members have been reported from the algal zone of coralloid roots:

Anabaena cycadae, Nostoc punctiforme, Oscillatoria, Azotobacter, Pseudomonas radicicola and even a few fungi. According to Kubitzki (1990) blue green algae or Cyanoba cteria (Anabaena, Nostoc and Calothrix) may rarely be present intracellularly (i.e. inside the cell) in the coralloid roots of Cycas. He opined that these algae fix nitrogen and promote the growth of host plant.

Due to the presence of blue-green algal members and some nitrogen-fixing bacteria, the function assigned to the coralloid roots is chiefly the nitrogen fixation. The presence and structure of endodermis, pericycle and vascular bundles in the coralloid roots are similar to that of normal roots. The xylem is exarch and triarch.

(iv) Stem:

Similar to root, the stem of Cycas also resembles internally with a dicotyledonous stem.

It shows the following anatomical features:

Epidermis is the outermost layer consisting of compactly arranged thick- walled cells. Presence of several persistent leaf bases makes the epidermis a discontinuous and ruptured layer. Cortex is large and consists of thin- walled, parenchymatous cells, filled densely with starch grains. It contains numerous mucilaginous canals and girdle traces.

Each mucilage canal is lined with many radially elongated epithelial or secretory cells (Fig. 8.19). Medullary rays connect the mucilage canals of the cortex with that of the pith Starch in the parenchymatous cells of the cortex is the source of ‘sago’. Endodermis and pericycle are not clearly demarcated.

Numerous vascular bundles remain arranged in a ring. The stele is ectophloic siphonostele. Each vascular bundle is conjoint, collateral, open and endarch (Fig. 8.20). The xylem consists of tracheids and xylem parenchyma (Fig. 8.21).

Protoxylem contains tracheids with spiral thickenings while the metaxylem has scalariform thickenings with bordered pits. Vessels are absent. The phloem is located outside the xylem and consists of sieve tubes and phloem parenchyma. Companion cells are absent.

Between the xylem and phloem lies the primary cambium, which remains active only for a short period. It is soon replaced by another ring of secondary cambium somewhere in the cortex. These successive cambial rings form 2-14 different vascular rings showing polyxylic condition in the old stem (Fig. 8.22).

Several broad and well-developed medullary rays are present between the vascular bundles. Pith is large, well-developed and parenchymatous. It contains many mucilaginous canals.

(v) Leaf Traces:

The leaf traces remain scattered in the cortical region of the stem and constitute the vascular supply to the leaves from the main vascular cylinder. Normally, there are four leaf traces which form the vascular supply to the leaf. Two of these are direct traces, while the remaining two axe girdle traces (Fig. 8.23).

The direct traces originate from the vascular cylinder lying in front of the leaf base while the girdle traces develop from the vascular cylinder lying opposite to that of direct traces. They proceed together and curve soon in opposite directions, and by girdling round the vascular cylinder they enter in the leaf base.

In the cortical region the girdle traces also remain connected with other leaf traces. At the time of their entrance in the petiole, the leaf trace bundles subdivide and form many petiole bundles. Such type of unique girdle traces of Cycas, which also occur in Magnoliaceae. show a close relationship of Cycadales of Gymnosperms and Magnoliaceae of dicotyledons.

(vi) Secondary Growth:

It is similar to that of dicotyledons. In the beginning, Cycas is monoxylic, i.e. contains a single ring of vascular bundles. But one or more concentric rings of vascular bundles appear outside the primary ring of bundles in the older stems showing polyxylic condition (Fig. 8.24)

By the activity of inter-fascicular and intra-fascicular cambia, which unite to form a cambium ring, the secondary growth is initiated. This cambium ring cuts secondary phloem towards outer side and secondary xylem towards inner side. Well-developed medullary rays traverse through the so-formed secondary vascular tissue.

After a short while this cambium ring stops functioning and a second cambium ring develops either in the parenchymatous cortex or in the region of pericycle This cambium ring also behaves in the similar fashion.

In this fashion, as many as 14 rings of vascular tissue may develop in the stem of Cycas pectinata of about 20 cm diameter showing polyxylic condition. Seward (1917) reported 12 such rings in the stem of C. media of about 30 cm diameter, and Schuster (1932) reported 22 such rings in the stem of C. rumphii having a diameter of about 85 cm.

Cambial rings towards the periphery of the stem form lesser number of vascular bundles. The cork cambium develops on the outer region of the cortex and cuts cork towards outer side and secondary cortex towards inner side.

(vii) Rachis:

The outline of transverse section is rhomboidal in the basal region of the rachis, biconvex in the middle cambium and roughly cylindrical at the tip region or at the apex of the rachis. Two arms of the bases of leaflets are present on the rachis, one on each side (Fig. 8.25).

In T.S. the rachis reveals the following structures from outside within:

Epidermis is the outermost layer of the rachis consisting of thick-walled cells. It is heavily circularized. On its upper as well as lower sides are present irregularly distributed sunken stomata. Hypodermis is present below the epidermis.

It is differentiated into outer 2-3 layers of chlorophyll-containing thin-walled cells of chlorenchyma and inner 4-6 layers of thick- walled lignified cells of sclerenchyma. Sclerenchyma is poorly-developed on the lateral sides. It is also seen intermixed with chlorenchyma.

Ground tissue is a large region consisting of thin- walled, parenchymatous cells. Many mucilaginous canals and vascular bundles are present in this region. The number and arrangement of mucilage canals have no definite relation with that of vascular bundles. Each mucilage canal is a double-layered structure consisting of an inner layer of epithelium cells surrounded by an outer layer.

Vascular bundles are arranged in the shape of an inverted Greek letter Omega (Ω) (Fig. 8.25). Towards the tip of the rachis the bundles are arranged in C-shaped manner and their number is comparatively less. Each vascular bundle remains surrounded by a bundle sheath (Fig. 8.26). It is conjoint, collateral and open.

The xylem in each vascular bundle is present towards inner side. It consists of tracheids and xylem parenchyma. Cambium separates the xylem from the phloem. Vessels are absent.

The vascular bundles are diploxylic, i.e. consists of two types of xylem viz. centripetal xylem and centrifugal xylem. Phloem, present towards the outer side of the vascular bundle, consists of sieve tubes and phloem parenchyma. Companion cells are absent.

The vascular bundles show different structure at different levels of rachis starting from the base up to the apex, especially with regard to their diploxylic nature.

Their brief description is under mentioned:

(a) Vascular Bundles At the Base of Rachis:

Only the centrifugal xylem is well-developed in the vascular bundles (Fig. 8.27A). Its protoxylem faces towards the centre showing endarch condition. Centripetal xylem is not developed.

(b) Vascular Bundles In the Middle of Rachis:

Both centripetal as well as centrifugal xylem are present showing diploxylic condition (Fig. 8.27B). Centripetal xylem is present just opposite to the protoxylem of the centrifugal xylem.

(c) Vascular Bundles At the Apex of Rachis:

Centripetal xylem is well-developed, triangular and exarch (Fig. 8.27C). Centrifugal xylem is much reduced and present in the form of two patches lying one on each side of the protoxylem elements of centripetal xylem. Centrifugal xylem is totally absent at the extreme tip of the rachis.

(viii) Leaflet:

Cycas leaflets are large, tough, thick and leathery. In a vertical section the leaflet is differentiated into a swollen midrib portion and two lateral wings (Figs. 8.28, 8.29). In C. revoluta and C. beddomei the wings are curved downward or revoluted at the margins but in C. circinalis, C. rumphii, C. pectinata and C. siamensis the margins are flat.

Epidermis is the outermost layer consisting of thick-walled cells. It is surrounded by a thick layer of cuticle. Upper epidermis is a continuous layer while the continuity of the lower epidermis is broken by many sunken stomata. On all the sides of the epidermal cells occur simple pits almost in regular series.

According to Pant and Mehra (1964), the stomata are of haplocheilic type (perigenous) in Cycas circinalis, C. revoluta and C rumphii. Hypodermis is sclerenchymatous and present below the epidermis. It is absent below the lower epidermis but in the midrib region it is several-celled thick.

Mesophyll is well-developed and remains differentiated into palisade and spongy parenchyma. A continuous layer of palisade is present below the sclerenchymatous hypodermis. Its cells are radially elongated and filled with chloroplasts. The palisade may be a continuous layer over the midrib as in Cycas beddomei, C. media, C. pectinata and C. revoluta, or it may be a discontinuous layer as in C. circinalis and C. rumphii.

Spongy parenchyma is present only in the wings, directly above the lower epidermis. Its cells are oval, filled with chloroplasts, and loosely arranged having many air-filled intercellular spaces. Transfusion tissue consists of two small groups of short and wide tracheid-like cells with reticulate thickenings or bordered pits on their walls.

These cells have been named as transfusion tissue by Von Mohl (1871), and were first described by Frank (1864). Few layers of transversely elongated cells are present in both the wings just in between the palisade and spongy parenchyma.

This represents the accessory transfusion tissue or secondary transfusion tissue. The secondary’ transfusion tissue has also been named as hydrostereom by Bernard (1904) or radial parenchyma by Pilger (1926). A great phylogenetic significance has been attributed to the transfusion tissue by Worsdell (1897).

Vascular bundle is one, and present in the midrib region of the leaflet. It is conjoint, collateral, open and diploxylic. The triangular centrifugal xylem is well-developed with endarch protoxylem. It is represented by two or sometimes more small groups on either side of the protoxylem.

Phloem is arc-shaped and remains separated by cambium. Phloem consists of sieve tubes and phloem parenchyma. Companion cells are absent. The portion of the midrib in between the palisade layer and lower hypodermal region is filled with parenchymatous cells. Some of these cells contain calcium oxalate crystals.

4. Reproduction in Cycas:

(i) Vegetative Reproduction:

The most common method of vegetative propagation in Cycas is by bulbils. The bulbils develop from the axil of the scaly leaves. They are more or less oval structures with a broad base narrowing towards the apex. Several scaly leaves are arranged spirally and compactly over a dormant stem in a bulbil (Fig. 8.30).

On detachment from the stem, a bulbil starts germination by producing many roots towards the lower side and a leaf towards the upper side. A bulbil from male plant will develop only into the male plant, while from the female plant will form only the female plant because Cycas is a strictly dioecious plant.

(ii) Sexual Reproduction:

Cycas is strictly dioecious, i.e. male and female sex organs are borne on separate plants. After several years of vegetative growth the plants start to form sex organs. Generally, Cycads of more than 10 years of age produce the sex organs.

The male plants develop male cones or male strobili bearing microsporophyll’s, while the female plants produce a loose collection of megasporophylls. The male cone is terminal while the megasporophylls are produced in succession with the leaves at the top of the stem.

Male Reproductive Structures:

The male cone (Fig. 8.31) or male strobilus is a large, conical or ovoid, compact, solitary and shortly-stalked structure, which is generally terminal in position. It sometimes attains a length of as much as 1.5 metre. In the centre of the cone is present a cone axis (Fig. 8.32).

Several perpendicularly attached microsporophyll’s are arranged around the cone axis in closely set spirals. At the base of male cone are present many young leaves. All the microsporophyll’s in a male cone are fertile except a few at its basal and apical parts.

The terminal growth of the stem is checked for sometime when a male cone appears at its apex. It is because of the fact that the apical meristem is used up during the development of the male cone. Cones of some species of Cycas are amongst the largest cones in the plant kingdom.

2. Microsporophyll’s, Microsporangia and Microspores:

Microsporophyll’s (Fig. 8.33) are flat, leaf-like, woody and brown-coloured structures with narrow base and expanded upper portion. The upper expanded portion becomes pointed and is called apophysis. Narrow base is attached to the cone axis with a short stalk.

Each microsporophyll contains two surfaces, i.e. an adaxial or upper surface and an abaxial or lower surface. On the adaxial surface is present a ridge-like projection in the middle and an apophysis at the apex (Fig. 8.33).

On the abaxial surface (Fig. 8.34A) are present thousands of microsporangia in the middle region in the groups of 3-5. Each such group is called a sorus. In between these groups are present many hair-like structures, which are very soft and one or two- celled structures (Fig. 8.34B).

In T.S. of a microsporophyll, there are present many microsporangia on the abaxial side (Fig. 8.35). Each shortly-stalked, oval or sac-like microsporangium is surrounded by 5-6 layers. The wall layers of each sporangium include an outer thick epidermis or exothecium, middle zone of thin-walled cells and an innermost layer of tapetum (Fig. 8.36).

Many pollen grains or microspores are present in each sporangium. In the expanded region of microsporophyll are present many mucilaginous canals and vascular bundles. Each sporangium is provided with a radial line of dehiscence, which helps in the dispersal of spores.

Microsporophyll’s are un-branched but Kashyap (1930) reported some abnormal branching of microsporophyll’s. On an average 700 (Cycas circinalis) to 1160 (C. media) sporangia per sporophyll have been reported. More than 7,00,00,00,000 microspores per cone may be present.

Each microspore or pollen grain is a rounded, unicellular and uninucleate structure surrounded by an outer thick exine and inner thin intine. Cytoplasm surrounds the centrally located nucleus. A large vacuole is also present (Fig. 8.37).

Scanning electron microscopic studies of Sahashi and Ueno (1986) on the pollen grains of Cycas revoluta suggest that they are oblong with 1-sulcate shrunken aperture. Reticulum-like sculpting’s are present on the inner layer of exine, and in this character Cycas resembles with Ginkgo biloba.

3. Development of Microsporangium:

It is of eusporangiate type (Fig. 8 .38). Few hypodermal sporangial initials divide penclinally to form outer primary wall cells and inner primary sporogenous cells. Primary wall cells divide and re-divide periclinally as well as anticlinally to form 5-7 cells thick wall of the sporangium while the primary sporogenous cells divide to form many sporogenous cells.

By further divisions the sporogenous cells develop into microspore mother cells. The latter divide reductionally to form haploid microspores or pollen grains arranged tetrahedrally.

The tapetum, which is utilized for the spore formation, develops either from the outermost layer of the sporogenous tissue or from the innermost layer of the wall tissue. Microspore is the first cell of the male gametophyte having haploid number of chromosomes.

The haploid chromosome number in Cycas is 11. But in C. revoluta it is also sometimes 12. The female plants are homogametic with XX-type of chromosomes while the male plants are heterogametic having chromosomes of XY-type.

Female Reproductive Organs:

True female cone or strobilus is absent Cycas. Female reproductive organs are present in the form of megasporophylls. Many megasporophylls are present around the apex of the monopodial trunk of the female plant above each crown of foliage and scaly leaves (Fig. 8.39).

Similar to foliage leaves, megasporophylls also remain spirally arranged at the apex of the stem but their number is very large and thus they appear like a rosette. Vegetative leaves and fertile megasporophylls are produced in an alternate succession without showing any effect on apical men stem.

Pant (1953) observed that usually the megasporophylls in Cycas are produced only once in a year. From the apex of the main stem the megasporophylls arise in an acropetal succession.

Each megasporophyll is considered a modification of foliage leaf. It reaches up to 30 cm or more in length in different species. It is a flat body consisting of an upper dissected or pinnate leafy portion, middle ovule-bearing portion and proximal petiole. Petiole varies in length in different species.

The middle part is comparatively wider than petiole and bears ovules arranged in two pinnate rows. The number of ovules varies between 2-12 in different species. The ovules are green when young but at maturity they are fleshy and bright orange or red-coloured structures.

The upper, conical sterile part of the megasporophyll is pinnately divided in Cycas revoluta (Fig. 8.40), C. pectinata (Fig. 8.41 B) and C. siamensis (Fig. 8.41 A). But the margin of the upper part is variously serrate with a tapering acute apex in C. beddomei (Fig. 8.42C), C. circinalis (Fig. 8.42A) and C. rumphii (Fig. 8.42B).

Cycas thouarsi contains the largest ovule amongst the living gymnosperms measuring about 7 cm in length. The megasporophylls remain covered by many yellow or brown-coloured hairs.

Cycas ovules are orthotropous, unitegmic and shortly-stalked. Generally, one or sometimes a few more ovules develop fully on a megasporophyll. Many un-pollinated ones remain small and ultimately abort.

Outer surface of the ovule may be smooth as in C. circinalis or covered with orange-yellow hairs as in C. revoluta. After fertilization these hairs are lost, the ovule changes into seed and its colour changes from orange-yellow to bright red.

The single integument is very thick and covers the ovule from all sides except a mouth-like opening called micropyle.

The integument consists of three layers:

(i) Outer, green or orange, fleshy layer called sarcotesta,

(ii) Middle, yellow, stony layer called sclerotesta, and

Several tannin cells and mucilage canals are present in the parenchymatous region of sarcotesta. Some pigments are also present in sarcotesta and epidermis. The sclerotesta consists of lignified thick-walled cells. The inner fleshy layer consists of parenchymatous cells, and it remains in close association with the nucellus.

The nucellus grows out into a beak-like portion called nucellar beak. The latter protrudes into the micropylar canal. Certain cells at the top of the nucellus dissolve and form a cavity like structure called pollen chamber (Fig. 8.43). Pollen grains are received in the pollen chamber after pollination.

The nucellus gets reduced in the form of a thin papery layer in mature seeds and encloses the massive female gametophyte (endosperm). An enlarged megaspore or the embryo-sac is present within the nucellus. The endosperm is formed by the repeated divisions of the megaspore nucleus followed by free cell formation.

Just below the pollen chamber is present an archegonial chamber. 3-6 archegonia are present in the female gametophyte near the archegonial chamber. The latter remains filled with a fluid.

3. Vascular Supply of the Ovule:

Stopes (1904) has worked on the vascular supply of Cycas seed. Out of several bundles of the megasporophyll only three enter the base of the ovule (Fig. 8.43). Out of these three bundles, the central one entefs into the base of the inner fleshy layer of the integument. After its entrance it divides into number of branches, all of which reach up to chalazal end of the nucellus. But none of them penetrates the nucellus.

Each of the remaining two lateral bundles enters the outer fleshy layer and bifurcates into a large outer branch and a small inner branch. The collateral and mesarch outer branch runs all through the outer fleshy layer up to the apex of the ovule. The remaining inner branch penetrates the strong middle stony layer and enters the inner fleshy layer, to which it supplies up to the micropylar end of the ovule.

4. Formation of Megaspores:

In the central region of the nucellus, the nucleus of one of the cell enlarges. Its cytoplasmic contents become dense and it also increases in its size. This cell represents the megaspore mother cell, which divides reductionally to form four haploid megaspores arranged in a linear tetrad (Fig. 8.44).

Out of these four megaspores, the upper three present towards the micropylar end degenerate, leaving only the lowermost functional megaspore or embryo sac cell. This is the fist cell of the female gametophyte.

5. Economic Importance of Cycas:

1. Cycas is used as a source of food in Japan, Australia, South East Asia, southern and eastern parts of India and some other countries. It is used in the preparation of starch and alcoholic drinks. The starch, extracted from its stem, is called ‘sago’.

‘Sago’ is prepared in the following way:

The bark of the trunk is removed, and the trunk is cut into thin discs. These are dried, ground and a paste is prepared by adding water Excess of water is added, and the paste is left for some time in a standstill position.

The starch settles down, and the clear upper liquid is drained off. Between the boards, the starch is rolled. This gives the starch a characteristic round shape. It is finally dried and sold as ‘sago’ in the market.

2. In Japan, seeds and stem of Cycas revoluta are used for preparing wine.

3. The juice obtained from young leaves of Cycas circinalis is used in skin diseases, vomiting of blood and stomach disorders.

4. The decoction of young red seeds of C. circinalis is used as a purgative and emetic.

5. To relieve the headache, giddiness and sore throat, the seeds of Cycas revoluta are prepared in the form of a tincture and used.

6. Cycas revoluta and C. circinalis plants are grown for ornamental purposes in various parts of the world.

7. The wood of Cycas revoluta is used for preparing small boxes and dishes.

8. Cycas leaves, being very large, are used for preparing baskets, mats, etc.

9. Cycas circinalis seeds are used in Democratic Kampuchea as a fish-poison.


Results

Identification and Characterization of a Novel Class of AMPs from HLB-Tolerant Citrus Relatives.

Through the comparative expression analysis of small RNAs and mRNAs between HLB-sensitive cultivars, HLB-tolerant citrus hybrid US-942 (Poncirus trifoliata × Citrus reticulata), and microcitrus Sydney hybrid 72 (Syd 72, Microcitrus virgate from M. australis × M. australasica) (14), we identified a list of candidate plant immune response genes that are potentially responsible for HLB tolerance. One candidate gene encodes a 67 amino acid (aa) peptide containing two predicted α-helix domains (15), which is homologous to a 109-aa Arabidopsis heat-stable protein HS1 with antimicrobial and antifungal activity (16). Here, we named this peptide SAMP.

To determine whether SAMP is associated with HLB tolerance, we cloned SAMP genes from HLB-tolerant citrus relatives, including the Australian finger lime (M. australasica), Australian desert lime (E. glauca), Hawaiian mock orange (Murraya paniculata), Khasi papeda (Citrus latipes), and seven trifoliate oranges (P. trifoliate). All of these citrus relatives have a long (109-aa) and at least one short (67-aa) version of SAMPs (SI Appendix, Figs. S1B and S2), whereas HLB-susceptible citrus varieties Citrus clementine (Cc) and Citrus sinensis (Cs) have only the long SAMP (LSAMP) with 118 aa and 109 aa in length, respectively, based on the Citrus Genome Database (https://www.citrusgenomedb.org) (SI Appendix, Fig. S2). SAMP has significantly higher mRNA levels in both HLB-tolerant hybrids US-942 and Syd 72 (SI Appendix, Fig. S1A) than LSAMP in the HLB-susceptible control trees. The SAMPs share high sequence similarity to the C-terminal domain of the LSAMPs. In finger lime (Ma), desert lime (Eg), trifoliate (Pt) Flying dragon, and Pt Florida, we identified two closely related SAMPs. Since the genome sequences of these citrus relatives are currently unavailable, there may be more SAMPs encoded in these genomes. We found that SAMP transcripts have a significantly higher mRNA expression level in HLB-tolerant varieties compared to LSAMP in the HLB-susceptible varieties (Fig. 1A).

A heat-stable antimicrobial peptide, SAMP, identified from HLB-tolerant citrus relatives has bactericidal activity. (A) The expression level of SAMPs in different citrus and citrus relatives was analyzed by qRT-PCR and normalized to Actin. The significant difference is indicated by *P < 0.05 analyzed by t test. (B) SAMPs were detected by Western blot analysis using an anti-MaSAMP antibody in phloem-rich tissue bark peels of Cs, Ma, and Pt. Ponceas staining (PS) was used as the loading control. (C and D) The bactericidal activity of different concentrations (C) or heat pretreatment (D) of MaSAMP solution was examined by Lcr viability/cytotoxicity assays. Streptomycin was used for comparative analysis. The buffer with 10 μM BSA was used as the control. The DMAO (green dye) and EthD-III (red dye) stains the live and dead bacteria, respectively.

Next, to probe if SAMP is present in phloem, where CLas is located, we generated a native antibody against SAMP and detected the 6.7-kD short version in the phloem-rich tissue-bark peels of HLB-tolerant Ma and Pt but not in the susceptible Cs (Fig. 1B). These results further support that the SAMPs are likely associated with the HLB-tolerance trait.

SAMP Has Bactericidal Activity and Is Heat Stable.

To test the ability of SAMPs to treat Liberibacter diseases, and to identify the most effective SAMP variant, we developed a rapid functional screening method, using a C. Liberibacter solanacearum (CLso)/potato psyllid/Nicotiana benthamiana interaction system to mimic the natural transmission and infection circuit of the HLB complex (14). We screened SAMPs from several citrus relatives and found that the SAMP from M. australasica Australian finger lime (MaSAMP) had the strongest effect on both suppressing CLso disease and inhibiting bacterial growth in plants (SI Appendix, Fig. S3), whereas the LSAMP has no or very weak effect. Thus, MaSAMP was used for the subsequent analyses.

To directly determine the bactericidal activity of MaSAMP on Liberbacter spp., we developed a viability/cytotoxicity assay of Liberibacter crescens (Lcr), a close culturable relative of the CLas and CLso (17 ⇓ –19). Using this assay, we found that 10 μM MaSAMP can kill the bacterial cells and induce aggregation as rapidly as 30 min after treatment (Fig. 1C). Lower MaSAMP concentrations of 1 μM or 100 nM can still kill the bacterium within 5 h after treatment (Fig. 1C). Furthermore, we found that MaSAMP, at a concentration as low as 100 nM, is more efficient at killing bacteria than the bactericidal antibiotic Streptomycin at a concentration as high as 172 μM (100 μg/mL) after 5 h of treatment (Fig. 1C). Streptomycin commonly needs longer time to kill the bacteria.

While the heat sensitivity of antibiotics is a major drawback for controlling CLas in citrus fields, we found that SAMPs are surprisingly heat stable. A prolonged exposure to extreme temperatures of 60 °C for 20 h had minimal effect on MaSAMP, which still retained most of its bactericidal activity (Fig. 1D), whereas Streptomycin completely lost its antibacterial activity following the same temperature incubation (Fig. 1D). Thus, SAMP is a heat stable plant-derived AMP that can directly kill Lcr and suppress CLso in plants.

SAMP Suppresses CLas in HLB-Positive Trees.

To determine whether MaSAMP can also suppress CLas in citrus trees, we used the pneumatic trunk injection method to deliver the MaSAMP solution into the HLB-positive citrus trees (SI Appendix, Fig. S4). In our first experiment, we obtained eight CLas-positive Citrus macrophylla with similar bacterial titer and disease symptoms for the treatment. Six trees were injected with MaSAMP (10 μM) and two trees were injected with the mock solution (Fig. 2). Eight wk after the first dose of MaSAMP injection, leaves were collected from the new flush on each tree for CLas titer analysis. The disease symptoms and the bacterial titer in all six treated trees were drastically reduced in comparison to the mock-treated plants (Fig. 2 AC). Furthermore, CLas was undetectable in one of the MaSAMP-treated trees (number 111, Fig. 2C). After the first two initial injections with 2 mo between the treatment, we monitored CLas titer on the trees without additional treatment for 6 mo and observed that the CLas titer started to increase after 5 mo, although the new leaves still looked healthy. CLas remained undetectable in tree 111. We then injected MaSAMP solution two more times (with 2 mo between treatments) and observed a rapid decrease in CLas titer (Fig. 2C). Moreover, new flush from the MaSAMP-treated plants displayed no HLB symptoms, whereas new flush from the mock-treated plants continued to display yellow striping symptoms (Fig. 2B). These results suggest that MaSAMP treatments at 2-mo intervals are effective to control HLB.

MaSAMP suppresses CLas in different HLB-positive citrus varieties. Citrus macrophylla (AC, after four injection doses), 'Madam Vinous' sweet orange (DF, after four injection doses), and 'Lisbon' Lemon (GI, after three injection doses) were injected with buffer (mock) or MaSAMP (10 μM). B, E, and H show new leaves or flushes in trees shown in A, D, and G, respectively. The treatment programs for A, D, and G were indicated in a timeline with months (M) at the upper of C, F, and I, respectively. The time points of injection (Inj, green arrow) and time of tree death (red star) are indicated. The CLas titer of individual trees in test A, D, and G at the indicated sampling time was measured by qPCR on CLas 16S rDNA using the USDA standard protocol (C, F, and I, Lower). Significant difference is indicated by *P < 0.01 analyzed by t test.

In our next round of testing, we used 14 HLB-positive 'Madam Vinous' sweet oranges (Cs) 10 mo after grafting inoculation. This set of trees had severe HLB declining symptoms with similar and high CLas titer. After MaSAMP treatments, the trees had increased growth and developed symptomless new flushes, while mock trees continued to exhibit symptomatic flushes (Fig. 2 D and E). Two mo after the first and fourth treatment, the CLas titer was examined. The titer was reduced after the first treatment and remained low after the fourth treatment in MaSAMP-treated trees, while it continued to increase in the mock-treated trees, two of which eventually died from HLB 4 mo after the last mock injection (Fig. 2F). In our third round of testing, we used seven HLB-positive 'Lisbon' Lemon trees with similar CLas titer. The two mock-treated trees were unable to produce new leaves and died 6 mo after the first injection, whereas the MaSAMP-treated trees had enhanced growth and exhibited healthy new flush (Fig. 2 G and H). Comparing the CLas titer 2 mo after first and third treatment, we found that the MaSAMP treatment could suppress the CLas growing in the lemon trees compared to the mock-treated trees (Fig. 2I). Taken together, these results demonstrate across three trials that SAMP injection can suppress CLas titer in three different HLB-susceptible citrus varieties and can cause trees in declining health to recover.

SAMP Treatment Safeguards Healthy Citrus Trees from CLas Infection.

Protecting HLB-negative citrus trees and saplings from CLas infection is critical for managing HLB. Establishment of defense priming in plants can promote faster and/or stronger host immune responses upon pathogen challenges (7, 8). To determine whether MaSAMP has priming activity, we applied it by foliar spray to Nb, tomato, and citrus plants. We found that MaSAMP application can induce the expression of a set of defense genes and activate systemic defense responses in Nb and tomato (SI Appendix, Fig. S5). Similarly, MaSAMP clearly triggered prolonged induction of defense response genes such as pathogenesis-related proteins PR1 and PR2 and an enzyme of SA biosynthesis and phenyl propanoid pathways, phenylalanine ammonia-lyase1 (PAL), in citrus trees (Fig. 3A) (7). Thus, SAMP can potentially “vaccinate” uninfected citrus trees and induce defense responses to combat against HLB and maybe other pathogen threats.

MaSAMP protects the healthy citrus trees from CLas infection. (A) The expression level of defense marker genes in the MaSAMP-treated 'Valencia' sweet orange trees was highly induced over a prolonged time course. The relative expression level was analyzed by qRT-PCR and normalized to Actin. (BE) Two sets of the 'Madam Vinous' sweet orange trees were foliar-sprayed with buffer (mock) or MaSAMP solution (10 μM) before ACP exposure (B) or graft infection (D). The treatment programs for B and D were indicated in a timeline with months (M) at the upper of C and E, respectively. The spray time points (green arrow) and the time when trees died (red star) were indicated. The CLas titer of individual trees in test B and D at the indicated sampling time was shown in the middle of C and E, respectively. The tables of the lower panel of C and E represent the number of the infected and dead trees. (F) MaSAMP was detected with Western blot by anti-MaSAMP antibody in the vascular fluid collected from MaSAMP-sprayed leaves. The midveins were tap-protected before spraying to avoid direct contact of MaSAMP with midveins. (G) Systemic leaves of trees that have lower leaves wiped with MaSAMP solution using a cotton ball were collected at 24 h or 7 d after MaSAMP application. The MaSAMP was detected by Western blot in the vascular fluid of the midvein from systemic untreated leaves. Ponceas staining (PS) was used as the loading control.

To identify the key signaling components involved in the SAMP-induced immune responses and to elucidate the underlying mechanism, we selected several master regulators of plant immune responses, including nonexpressor of pathogenesis-related gene 1 (NPR1) (20), suppressor of G2 allele of skp1 (SGT1) (21), and the coreceptor of several receptor-like kinases involved in plant defense, BRI1-associated receptor kinase1 (BAK1)/somatic embryogenesis receptor kinase3 (SERK3) (22), to assess their role in SAMP-triggered immunity. We performed virus-induced gene silencing (VIGS) (23) to knock down these immune regulators in Nb plants and then examined the PR gene expression after MaSAMP treatment (SI Appendix, Fig. S6). The results showed that silencing of NPR1 and SGT1 largely abolished SAMP-induced PR gene expression (SI Appendix, Fig. S6A), indicating that SAMP-triggered plant immunity is NPR1- and SGT1-dependent. However, VIGS of both BAK1 homologs SERK3A and SERK3B in Nb didn’t have an effect, suggesting that BAK1/SERK3 are not required for SAMP recognition and signaling.

To test the protection ability of SAMP on citrus trees, we applied the MaSAMP solution or buffer as mock treatment by foliar spray onto 20 young healthy 'Madam Vinous' sweet orange trees. At 5 d after treatment, the trees were exposed to ACP carrying CLas under the “no choice feeding” condition for 21 d. We subsequently treated trees with MaSAMP solution by foliar spray every 2 mo. At 12 mo after inoculation, the trees sprayed with MaSAMP exhibited enhanced growth compared to the mock-treated trees (Fig. 3B). At 14 mo after inoculation, 9 of 10 mock-treated trees tested CLas positive and 4 died (Fig. 3C). In the MaSAMP-treated trees, only three tested positive, each with a very low CLas titer (Fig. 3C). For the next trial, we grafted HLB-positive budwood as inoculum onto nine MaSAMP-treated and nine mock-treated HLB-negative rootstock trees. Similar to the ACP-mediated inoculation, the MaSAMP-treated trees had enhanced growth compared to the mock treatment (Fig. 3D). At 10 mo post grafting, all of the mock-treated trees were HLB positive, whereas only four of nine MaSAMP-treated trees were HLB positive, with significantly low CLas titer (Fig. 3E).

Foliar-spray delivery is a common and practical method for field applications. To determine whether SAMP enters the citrus vascular system following foliar application, we cut out the covered midvein from leaves after spray application and collected the vascular fluid. We detected vascular SAMP uptake as early as 6 h post-spray (Fig. 3F). To eliminate potential mist contamination, we next manually wiped MaSAMP solution with cotton balls onto the lower leaves of the citrus tree and collected the vascular fluid from the midvein of upper untreated leaves. We detected MaSAMP was systemically transported to the upper leaves as early as 24 h post-treatment, and the transported MaSAMP remained stable in vasculature for at least 7 d (Fig. 3G). About 9.8 to 22 μM of MaSAMP was detected in the vascular fluid collected from leaf midribs at 24 h post-treatment (SI Appendix, Fig. S7A), which we found to be a sufficient concentration to rapidly kill Lcr in the viability assay (SI Appendix, Fig. S8). In addition, the amount of MaSAMP detected in the citrus leaves was negatively correlated with the CLas titer in the leaves, which further supports that MaSAMP inhibits CLas (SI Appendix, Fig. S7B). Thus, SAMP is rapidly taken up by citrus leaves and moves systemically in the trees through vasculature, where CLas is present. The existence of SAMP in the vascular system is long lasting.

SAMP Is Effective Against α-Proteobacteria and Causes Cell Leakage and Lysis.

Many AMPs exhibit antimicrobial activity across an array of microorganisms (24). To better understand the range of SAMP’s antimicrobial activity, we performed viability assays on a variety of bacteria including gram-positive Bacillus subtillis (Bs), gram-negative α-proteobacteria including Lcr and Agrobacterium tumefaciens (At), and γ-proteobacteria including Escherichia coli and Xanthomonas campestris pv. Vesicatoria (Xcv). MaSAMP has strong antibacterial activity against Lcr and At at 10 μM but not against Bs, E. coli, or Xcv (Fig. 4A). The minimum inhibitory concentration of MaSAMP to inhibit Lcr and At is about 10 μM (SI Appendix, Fig. S8). High MaSAMP concentrations of 200 μM and 120 μM were needed to significantly inhibit Bs and E. coli, respectively (Fig. 4A). With the limited number of bacteria strains tested, we found that SAMP may be more effective on α-proteobacteria.

MaSAMP is most effective on α-proteobacteria. (A) Bacteria viability/cytotoxicity assays of MaSAMP were performed on Bacillus subtilis (Bs), Liberibacter crescens (Lcr), Agrobacterium tumefaciens (At), Escherichia coli (E. coli), and Xanthomonas campestris pv. Vesicatoria (Xcv). The green and red cells indicate the live and dead cells, respectively. Pictures were taken at 5 h post-treatment. (B) TEM image of Lcr cells treated with 10 μM MaSAMP or BSA (mock) showed cytosol leakage and vesicle releasing 0.5 h post-treatment. Cell lysis was observed at 2 h post-treatment. Cytosol leakage or vesicle release are indicated by the black arrows.

To understand the mechanism of MaSAMP bactericidal activity, morphological changes of Lcr after MaSAMP treatment were observed using transmission electron microscopy. Application of 10 μM MaSAMP to Lcr caused cytosol leakage and the release of small extracellular vesicles after 30 min of incubation (Fig. 4B). The Lcr cells were lysed within 2 h of incubation. Vesicle release was potentially caused by compromised maintenance of membrane lipid asymmetry, induced lipopolysaccharide modifications, or accumulation of misfolded proteins in the outer membrane (25). We isolated the membrane fraction from the MaSAMP-treated Lcr and detected the enrichment of MaSAMP in the outer membrane fraction as compared with the inner membrane fraction (SI Appendix, Fig. S9). Thus, MaSAMP likely disrupts mainly the outer membrane of Lcr and breaks the bacterial cells, which leads to cell lysis.

The Second α-Helix of SAMP Is the Major Bactericidal Motif.

To understand the mechanism of action of SAMP, we modeled its structure, which contains two short α-helical fragments connected by a proline hinge region with a loose N and C terminus (Fig. 5 A and B) (26). The amphipathic helix2 has the hydrophobic residues facing one side (Fig. 5C). We detected that MaSAMP forms polymers (probably hexamers based on the molecular weight) in the native gel (Fig. 5D), suggesting that this peptide likely forms a pore-like structure. Different sodium dodecyl sufate (SDS)-resistant MaSAMP oligomers were observed in the SDS denaturing gel (Fig. 5E), indicating that the oligomers of SAMP are rather stable. To determine the critical domain of SAMP for its function, we generated a series of truncated versions of MaSAMP, including the double-helix hairpin (MaSAMPNC), α-helix1 (MaSAMP-helix1), and α-helix2 only (MaSAMP-helix 2), to test their bactericidal activity. The results indicate that α-helix2 is largely responsible for the antibacterial activity, though full-length MaSAMP activity is slightly higher (Fig. 5 F and G). By tripling the amount, MaSAMP-helix2 can reach up to 90% activity of the full-length MaSAMP (Fig. 5G and SI Appendix, Fig. S10). Furthermore, we also detected the polymers (again most likely hexamers based on the molecular weight) using only the helix2 domain (MaSAMP-helix2) (Fig. 5D), further suggesting that this peptide forms oligomers using its helix2 domain.

The α-helix2 domain of MaSAMP is the key bactericidal motif, and SAMP is present in fruits and rapidly degraded by pepsin. (A) The diagram of the SAMP structure. (B) The predicted structure of SAMP by the SWISS-MODEL. The hydrophobic residues are marked in red. (C) The helical wheel diagram of the α-helix2 domain was predicted. The hydrophobic residues are circled in blue. (D) MaSAMP and MaSAMP-helix2 domain form only polymers (likely hexamers) in the native PAGE gel. (E) MaSAMP forms SDS-resistant oligomers. (F and G) The bactericidal activity of various truncated MaSAMPs was examined using Lcr viability/cytotoxic assay. The green and red cells indicate the live and dead cells, respectively. (H) MaSAMP phytotoxicity was assessed by infiltrating different concentrations of MaSAMP or BSA solution into the leaf of sweet orange. (I) MaSAMP was detected by Western blot using the anti-MaSAMP antibody in the fruit tissue of Australian finger lime (Ma) and trifoliate orange (Pt) but not Lemon (Cl). The corresponding fruit pictures are shown in the upper panel. (J) MaSAMP was rapidly degraded after incubation with human pepsin over a time course.

Toxicity Assessment of SAMP.

Because SAMP is internalized by citrus, it is important to test its phytotoxicity. We injected different concentrations of MaSAMP solution directly into citrus leaves and found that MaSAMP has little phytotoxicity even at a concentration as high as 100 μM (Fig. 5H). Furthermore, we found that MaSAMP can be detected in fruit tissue of both HLB-tolerant Australian finger lime and trifoliate orange by Western blot analysis (Fig. 5I). MaSAMP is very sensitive to human endopeptidase Pepsin, a major gastric enzyme produced by stomach chief cells (Fig. 5J). Thus, MaSAMP in Australian finger lime has already been consumed by humans for hundreds of years and can be easily digested. These results suggest a low possibility of toxicity of SAMP on citrus and humans, although additional safety assessment tests are necessary for regulatory approval.


Use and Management

Although a rather large tree, tulip-poplar could be used along residential streets with very large lots and plenty of soil for root growth if set back 10 or 15 feet. Not generally planted in large numbers and probably best for a specimen or for lining commercial entrances with lots of soil space. Trees can be planted from containers at any time in the south but transplanting from a field nursery should be done in spring, followed by faithful watering. Plants prefer well-drained, acid soil. Drought conditions in summer can cause premature defoliation of interior leaves which turn bright yellow and fall to the ground, especially on newly-transplanted trees. The tree may be short-lived in parts of USDA hardiness zone 9, although there are a number of young specimens about two feet in diameter in the southern part of USDA hardiness zone 8b. It is usually recommended only for moist sites in many parts of Texas, including Dallas, but has grown in an open area with plenty of soil space for root expansion near Auburn and Charlotte without irrigation where the trees are vigorous and look nice.

There are several cultivars: `Aureo-maculatum' -- leaves with yellow blotches, `Aureo-marginatum' -- leaves edged with yellow, `Fastigiatum' -- columnar growth, `Pyramidale' -- narrow growth habit. None are commonly available.

Pests

Aphids, particularly Tuliptree aphid, can build up to large numbers, leaving heavy deposits of honeydew on lower leaves, cars, and other hard surfaces below. A black, sooty mold may grow on the honeydew. Although this does little permanent damage to the tree, the honeydew and sooty mold can be annoying.

Tuliptree scales are brown, oval and may be first seen on lower branches. Scales deposit honeydew which supports the growth of sooty mold. Use horticultural oil sprays in spring before plant growth begins.

Tuliptree is considered resistant to gypsy moth.

Diseases

Tuliptree is attacked by several cankers. Infected, girdled branches dieback from the tip to the point of infection. Keep trees healthy and prune out infected branches.

Leaf spots are usually not serious enough to warrant chemical controls. Once leaves are heavily infected the opportunity for chemical control is lost. Rake up and dispose of infected leaves. Leaves often fall during summer and litter the ground with yellow, spotted leaves.

Powdery mildew causes a white coating on the leaves and is not usually harmful.

Sooty mold makes a black coating on leaves and stems. The fungus grows on the honeydew left by insects, particularly aphids. Control sooty mold by preventing the build up of insects populations.

Verticillium wilt causes wilting and death of leaves on infected branches. Severe infections kill trees. Keep trees vigorous with a regular maintenance program, including fertilizer.

During hot, dry weather interior leaves turn yellow and fall off. This condition is due to the weather and is not a disease. The problem is most common on newly transplanted trees, but also develops frequently on established trees. Yellowing may be preceded by small, angular, brown spots on the leaves.


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