Is it possible to accelerate active transport in plant vascular system using some electronic method?

Is it possible to accelerate active transport in plant vascular system using some electronic method?

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I am from electronics background and got an understanding that electrical current affect plant growth. Based on this phenomenon, I got an idea but before I formulate my hypothesis and start experimentation around it, I am looking for some expert opinion on this.

Can we accelerate mineral transport like water movement from root to leaf by some external influences?

46 Q&As for Learning Plant Physiology

Plants need to carry out gas exchange because they use aerobic cellular respiration (like animals). As a result, they need to obtain molecular oxygen and release carbon dioxide. In addition to aerobic cellular respiration, plants also need to obtain carbon dioxide to carry out photosynthesis and to release the molecular oxygen that is the product of this reaction.

More Bite-Sized Q&As Below

2. What are the main gas exchange organs in plants? How does this process take place?

In the covering of the leaves and of the primary structure of the stem, gas exchange is carried out through the cuticle and pores of the epidermis. In the covering of the secondary structure of the stem of woody plants, gas exchange is carried out through the lenticels of the periderm (small breaches in cork). Gas exchange in plants is carried out via simple diffusion. 

Plant Transpiration and Stomata

3. What is plant transpiration? What are the two main types of plant transpiration processes? Which of them has a greater volume?

Transpiration is the loss of water from the plant to the atmosphere into the form of vapor.

Transpiration occurs through the cuticle of the epidermis (cuticular transpiration) or through the ostioles of the stomata (stomatal transpiration). The most important of the two is stomatal transpiration, since it is more intense and is physiologically regulated. 

4. What are stomata? How do these structures participate in plant transpiration?

Stomata (singular, stoma) are small specialized passageways for water and gases present in the epidermis of plants. As the plant needs to lose more or less water and heat, the stomata respectively close or open, preventing or allowing the movement of gases via diffusion. 

5. What elements compose stomata?

A stoma is made of a central opening, called the ostiole, or slit, surrounded by two guard cells responsible for closing and opening. A substomatal chamber is located under the ostiole.

6. How do plants control the opening and the closing of stomata?

The opening and the closing of stomata depend upon the plant's need to lose water and heat through transpiration (the exit of water vapor means the elimination of heat). When the plant has excessive, water the guard cells become turgid and the ostiole opens. When little water is available, the guard cells become flaccid and the ostiole closes.

Water enters and exits stomata via osmosis.

Other factors such as light intensity and carbon dioxide concentration in the leaves influence the opening and the closing of stomata. When luminosity is high the photosynthesis rate increases and the stomata open to absorb more carbon dioxide from the environment and release heat when luminosity is low, stomata tend to close. When the carbon dioxide concentration in the photosynthetic parenchyma is low, stomata open to absorb more of the gas to make photosynthesis possible when its concentration is high, stomata tend to close.

7. Do the stomata of plants placed in a dryer than usual environment remain open for more or less time?

If plants from a moister region are transferred to a drier region, it is likely that their stomata will remain closed for a longer time, because the time during which stomata are open will be reduced to lower the loss of water via transpiration.

8. Why do some plants adapted to a dry environment open their stomata only at night?

During the day in dry habitats, guard cells become flaccid and stomata close as a result, carbon dioxide is unable to move along to participate in diurnal photosynthesis. Some plants from dry regions solve this problem through the method of nocturnal carbon dioxide fixation. At night, when water loss by transpiration is lower, the stomata open, carbon dioxide enters and it is stored within parenchymal tissues. During the day the stored gas is mobilized to be used in photosynthesis.

9. How has the position of stomata changed in some plants to prevent excessive water loss via transpiration?

In some plants whose leaves receive too much sunlight, stomata concentrate in the inferior epidermis. As a result, they contain  less heat, and less water is lost via stomatal transpiration. In other plants adapted to dry environments, the stomata group in certain regions of the leaf, as over the surface of these areas, the water concentration of the air is higher compared to in the environment and the loss of water via transpiration is thus reduced. Some plants from dry climates also have stomata within cavities.

10. Is transpiration the only way through which leaves lose water?

Plants do not only lose water in the form of vapor, as is the case in transpiration. Leaves also lose liquid water through a phenomenon known as guttation. Guttation takes place through structures called hydathodes, which are similar to stomata. Guttation mainly occurs when transpiration is difficult due to high air humidity or when the plant is placed in watery soil. 

11. When air humidity is high, does the transpiration of a plant increase or decrease?

When air humidity is high, transpiration decreases. Since transpiration is a simple diffusion process, it depends on the concentration gradient of water between the plant and the environment. If the atmosphere has too much water vapor, the gradient becomes low or even reversed. 

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Plant Transport

12. How do the volume of water absorption and the volume of water transpiration vary in plants over the course of a day? Overall, how can these quantities be compared?

During the day, the volume of water transpired is higher than the volume absorbed by the roots. At night, the situation reverses and the roots absorb more water than the volume of water transpired.

It can be observed that the volume of water transpired and the volume of water absorbed practically equal over the course of a day. 

13. How do plants solve the problem of transporting substances throughout their tissues?

In bryophytes, substance transport is carried out by diffusion. Tracheophytes (pteridophytes, gymnosperms and angiosperms) contain specialized conducting vessels: xylem, which carries water and mineral salts, and phloem, which transports organic materials (sugar).

14. Is the transportation of gases in tracheophytes carried out through vascular tissues?

Carbon dioxide and oxygen are not transported through xylem or phloem. These gases reach the cells and exit the plant via diffusion through intercellular spaces or between neighboring cells. 

15. Are xylem and phloem made of living cells?

The cells that constitute xylem ducts are dead cells killed by lignin deposition. Phloem cells are living cells. 

16. What is the importance of lignin in xylem formation?

Lignin is important because it is deposited on the cell wall of xylem cells, providing impermeability and rigidity to xylem vessels.

17. What is root pressure?

Root pressure is the pressure that forces water from the soil to be absorbed by xylem in the root. It is caused by the osmotic gradient between the interior of the root and the soil.

18. What is capillarity? How is this phenomenon chemically explained? What is the relevance of capillarity for water transport in plants?

Capillarity is the phenomenon through which water moves inside extremely thin tubes (capillaries) aided by the attraction force between water molecules and the capillary wall. The phenomenon of capillarity is possible because water is a polar molecule that forms intermolecular hydrogen bonds. Therefore, there is an electrical attraction (adhesion force) between the capillary wall and the water molecules, which then pull each other (cohesion force), since they are bound. Other liquids may also move inside capillaries via capillarity, and not just water.

Capillarity is not particularly relevant for the transport of water in plants. It only contributes to a few centimeters of ascent.

19. What forces cause water to flow from the roots to the leaves within xylem?

Water enters the roots due to the root pressure and a water column is maintained within xylem from the roots to the leaves. The most important factor that makes water go up is transpiration, mainly in the leaves. As the leaves lose water via transpiration, their cells tend to attract more water, creating suction inside xylem. The cohesion property of water that keeps its molecules bound (one pulls the other) by hydrogen bonds helps in the process.

20. What is tree girdling? What happens to a plant when the girdle is removed from the stem (below the branches)?

Malpighi’s girdling, or tree girdling, is the removal of a complete external girdle containing the phloem (which is more external)ਏrom a stem, all the while preserving the xylem (which is more internal).

When a girdle is removed below the branches like that, the plant dies because organic food (sugar) is unable to move into the region below the girdle and, as a result, the roots die from the lack of nutrients. When the roots die, the plant does not obtain water or mineral salts and dies as a result.

Plant Hormones

21. What are plant hormones?

Plant hormones, also called phytohormones, are substances that control embryonic development and growth in adult plants. 

22. What are the main natural plant hormones and what are their respective effects?

The main natural plant hormones and their respective effects are the following:

Auxins (the best known natural auxin is IAA, indoleacetic acid): their function is to promote plant growth, distension and cellular differentiation. Gibberellins: their effect is similar to that of auxins (growth and distension) they stimulate flowering and fruit formation and activate seed germination. Cytokinins: they increase the cellular division rate and, together with auxins, help growth and tissue differentiation and slow the plant aging process. Ethylene (ethene): this is a gas released by plants, which participates in the growth process and has a noteworthy role in ripeningਏruitਊnd leaf abscission.

23. What is the coleoptile? Why does the removal of the extremity of the coleoptile prohibit plant growth?

The coleoptile is the first (one or more) aerial structure of the sprouting plant that emerges from the seed. It encloses the young stem and the first leaves, protecting them.

The top of the coleoptile is generally the region where auxins are produced. If this region is removed, plant growth stops, since auxins are necessary to promote growth and tissue differentiation.

24. What is indolacetic acid (IAA)?

Indolacetic acid (indolyl-3-acetic acid), or IAA, is the main natural auxin produced by plants. It promotes plant growth and cellular differentiation.

25. What are synthetic auxins and what are their uses?

Synthetic auxins, such as indolebutyric acid (IBA) and naphthalenic acid (NAA), are substances similar to IAA (a natural auxin) but which are artificially produced. Some are used to accelerate methods of asexual reproduction (such as grafting or budding) and others are even used as herbicides since they selectively kill some plants (mainly dicots).

26. Where is a large amount of IAA found in plants?

Auxins are produced and found in large amounts in the apical buds of the stem and shoots as well as in young leaves.

27. How do phytohormones help the development of parthenocarpic fruits?

Parthenocarpic fruits are those produced without fertilization. Some plants produce parthenocarpic fruits naturally, such as the banana tree, stimulated by their own hormones.

Angiosperms that do not naturally produce parthenocarpic fruits may do so if auxins are applied to flowers before fertilization. Therefore, even without fertilization, the ovaries grow and fruits are formed, although they are seedless. 

29. What happens when the auxin concentration in certain structures of the plant is over that of the action range of the hormone?

In some parts of the plant (the stem, roots, lateral buds), there are auxin concentration ranges in which the hormonal action is positive (it stimulates growth). It has been observed that concentrations over the upper limit of those ranges have the opposite effect (the inhibition of growth).

30. What is the phenomenon of apical dominance in plants? How can it be artificially eliminated?

Apical dominance is the phenomenon through which high (over the positive range limit) auxin concentrations due to auxins from the apical bud moving down the stem inhibit the growth of the lateral buds of the plant. At the beginning of stem development, apical dominance causes plant growth to be longitudinal (upwards), since the growth of lateral buds remains inhibited. As the lateral buds become more distant from the apex, the auxin concentration in these buds lowers and shoots grow more easily.

The growth of tree branches can be stimulated by preventing apical dominance through the removal of the apical bud.

31. What are gibberellins? Where are they produced?

Gibberellins are plant hormones that stimulate plant growth, flowering and fruit formation (also parthenocarpy) and the germination of seeds. There are more than 70 known types of gibberellins. Gibberellins are produced in the apical buds and young leaves.

32. What are cytokinins? Where are they produced?

Cytokinins are phytohormones active in the promotion of cellular division. They also slow down the aging of tissues and act together with auxins to stimulate plant growth. Cytokinins are produced by the root meristem and are distributed through the xylem.

33. What plant hormone is remarkable its ability to stimulate flowering and fruit ripening? What are the uses and practical setbacks of this hormone?

The plant hormone notable for its ability to stimulate and accelerate fruit ripening is the gas ethylene (ethene). Because it is a gas, ethylene acts not only in the plant that produces it but also in neighboring ones.

Some fruit processing industries use ethylene to accelerate the ripening of fruit. On the other hand, if the intensification or acceleration of fruit ripening is not desirable, care must be taken to prevent mixing of ripe fruits that release ethylene with others.

Plant Tropisms

34. Are the development and growth of plants only influenced by plant hormones?

Physical and chemical environmental factors, such as intensity and position of light in relation to the plant, gravitational force, temperature, mechanical pressures and the chemical composition of the soil and of the atmosphere, can also influence the growth and development of plants.

35. What are plant tropisms?

Tropisms are movements caused by external stimuli. In botany, the plant tropisms studiedਊre: phototropism (tropism in response to light), geotropism (tropism in response to the gravity of earth) and thigmotropism (tropism in response to mechanical stimuli).

36. In which direction does the growth of one side of a stem, branch or root cause the overall structure to curve?

Whenever one side of a stem, branch or root grows more than the other side the structure curves towards the side that grows less. (This is an important concept for plant tropism problems.)

37. What is phototropism?

Phototropism is the movement of plant structures in response to light. Phototropism may be positive or negative. Positive phototropism is when the plant movement (or growth) is towards the light source and negative phototropism is when the movement (or growth) is opposite, moving away from the light source.

Phototropism is related to auxins since the exposure of one side of the plant to light makes these hormones concentrate in the darker side. This causes the effect of auxins on the stem to be positive, meaning that the growth of the darker side is more intense and the plant arcs towards the lighter side. In roots, (when subject to light, in general and experimentally) the effect of auxins is negative (over the positive range), the growth of the darker side is inhibited, and the root curves towards that side.

38. What are the types of plant geotropisms? Why do the stem and the roots present opposite geotropisms?

The types of geotropisms are positive geotropism, in which the plant grows in favor of gravitational force, such as in roots, and negative geotropism, which is against gravitational force, such as in the stem.

Root geotropism and stem geotropism are opposite due to the different sensitivities to auxin concentrations in these structures. The following experiment can demonstrate the phenomenon: Stems and roots are placed in a horizontal position (parallel to the ground) and auxins naturally਌oncentrate along their bottom part. Under this condition, we can observe that the stem grows upwards and the root grows downwards. This happens because, in the stem, the high auxin concentration in the bottom makes that side grow (longitudinally) more and the structure arcs upwards. In the root, the high auxin concentration in the bottom inhibits the growth of that side and the upper side grows more, making the root curve downwards.

39. What is thigmotropism?

Thigmotropism is the movement or growth of a plant in response to mechanical stimuli (touch or physical contact), such as when a plant grows around a supporting rod. This occurs in grape and passionfruit vines, for example.


40. What is a photoperiod?

A photoperiod is the daily time period of light exposure of a living organism. The photoperiod may vary according to the time of the year.

41. What is photoperiodism?

Photoperiodism is the biological response of certain living organisms to their daily amount of light exposure (photoperiod).

42. What plant organs are responsible for the perception of variations in light? What pigment is responsible for this perception?

Leaves are mainly responsible for the perception of light intensity in plants. The pigment that is able to perceive light variations, and which controls photoperiodism, is called phytochrome.

43. How does photoperiodism affect the flowering of some plants?

Flowering is a typical and easy to observe example of photoperiodism. Most flowering plants flower only during specific periods of the year or when placed under certain conditions of daily illumination. This occurs because their blossoming depends on the duration of the photoperiod, which in turn varies with the season of the year. Flowering is also affected by exposure to certain temperatures.

44. What is the critical photoperiod? How can the critical photoperiod of flowering be experimentally determined?

The critical photoperiod is the limit of the duration of the photoperiod after which some biological response occurs. This limit can be a maximum or a minimum, depending on the characteristics of the biological response and to the studied plant.

To determine the critical photoperiod of flowering, 24 groups of plants of the same species can be used and the following experiment can be carried out: Each group is subject to a different photoperiod: the first group receives 1 hour of daily exposure to light the second 2 hours the third 3 hours and so on, until the last group is exposed to 24 hours. We can observe that beyond a specific duration of light exposure, plants present or do not present flowering, and the remainder submitted to a shorter photoperiod present the opposite behavior. The duration of the light exposure that separates these two groups is the critical photoperiod.

45. How can plants be classified according to their photoperiodism-based flowering?

According to their photoperiodism-based flowering, plants can be classified as: long-day plants, which depend on longer photoperiods than the critical photoperiod to flower as short-day plants, which depend on shorter photoperiods than the critical photoperiod to flower and as indifferent plants, whose flowering does not depend on the photoperiod.


46. Why do most plants present opposite phyllotaxis?

Phyllotaxis is the way leaves are arranged along shoots. Most plants have opposite phyllotaxis (alternating in sequence, one on one side of the shoot, the following on the opposite side) as a solution to prevent leaves from blocking the sun received by other leaves, thus improving the efficiency of photosynthesis.

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XYLEM INTERMIXED WITH PHLOEM1, a leucine-rich repeat receptor-like kinase required for stem growth and vascular development in Arabidopsis thaliana

The regulation of cell specification in plants is particularly important in vascular development. The vascular system is comprised two differentiated tissue types, the xylem and phloem, which form conductive elements for the transport of water, nutrients and signaling molecules. A meristematic layer, the procambium, is located between these two differentiated cell types and divides to initiate vascular growth. We report the identification of a receptor-like kinase (RLK) that is expressed in the vasculature. Histochemical analyses of mutants in this kinase display an aberrant accumulation of highly lignified cells, typical of xylem or fiber cells, within the phloem. In addition, phloem cells are sometimes located adjacent to xylem cells in these mutants. We, therefore, named this RLK XYLEM INTERMIXED WITH PHLOEM 1 (XIP1). Analyses of longitudinal profiles of xip1 mutant stems show malformed cell files, indicating defects in oriented cell divisions or cell morphology. We propose that XIP1 prevents ectopic lignification in phloem cells and is necessary to maintain the organization of cell files or cell morphology in conductive elements.

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Pollen tube growth and 1st sampling stage

The characterization of pollen tube growth and its visual developmental symptoms in spinach female flowers were conducted to identify the best time point for 1st stage RNA-seq library construction. The aim was to find metabolic pathways involved in very early pollination-induced developmental changes within sepal after pollination. As sepals grow with the developing seeds after pollination, so time point (1st stage of sepal development) just before fertilization was desired. For this, pollen tube growth was measured at various times 0, 10, 12, 14 h after pollination (HAP). It was observed that pollen tube maintained a relatively steady, non-linear growth, reached at the end of ovule after 12 HAP but before 14 HAP (Figure S1C). So, 12 HAP was considered the 1st time point for library construction.

Transcriptome assembly and differentially expressed genes analysis

To reveal the alternation in gene expression during sepal development after pollination, the non-strand-specific cDNA libraries were constructed from Cornel-9 unpollinated (UNP) and pollinated (12, 48, and 96 HAP) flower sepals with three biological replicates. A total of 148,241,329 paired-end clean reads that were 150 bp in length from 12 libraries were generated. 12 to 14.1 million clean reads per library from unpollinated and 9 to 14.8 million clean reads per library from pollinated flower sepals were generated. Clean reads were mapped to the spinach draft genome [16]. The mapping rate was over 91% for samples of each stage (Table S1). Differential expression analysis was conducted by a continuous comparison system to determine the differentially expressed genes at each stage after pollination. A total of 2825 genes were expressed differentially between unpollinated (UNP) and 12 HAP with 1443 upregulated and 1382 downregulated genes, decreased to 1782 between 12 HAP and 48 HAP with 715 upregulated and 1067 downregulated genes, and 1061 between 48 and 96HAP with 696 upregulated and 366 downregulated genes. The summary of DEGs in all designed comparisons is reported in Fig. 1a. Variability among the replicates of each treatment for DEGs is presented by a hierarchical heatmap in Fig. 1c.

a Unique and shared differential expression of unigenes in UNP vs 12HAP, 12 vs 48HAP, and 48 vs 96HAP pairwise analysis. b Representatives sepal phenotypes at different stages including Unpollinated flower sepal (UNP), sepal at 12 h after pollination, before fertilization (12HAP), sepal at 48 h after pollination (48HAP), and sepal with early visible symptoms of development at 96 h after pollination (96HAP). c Hierarchical heatmap showing the variability among replicates of each treatment for DEGs

KEGG and GO enrichment analysis

To further confirm the functional annotation, we performed the KEGG enrichment analysis for differentially expressed genes at each selected time point (Figure S2). The results suggested that the ‘plant phytohormone signal transduction’ was the significantly enriched pathway in all comparisons, while the other pathways such as ‘Tryptophan metabolism’, ‘DNA replication’, ‘Glycin, serine, threonine metabolism’, and ‘Valine, leucine and isoleucine degradation’ pathways were overrepresented at UNP vs 12 HAP. Pathways related to ‘glycan degradation’, ‘carbon metabolism’ at 12 vs 48HAP and ‘Phenylpropanoid biosynthesis’, ‘Flavone and flavonol biosynthesis pathways’ and ‘alpha-Linolenic acid metabolism’ were enriched at 48 vs 96HAP. Additionally, ‘secondary metabolites biosynthesis pathway’ was of significant enrichment at 12 vs 48HAP and 48 vs 96HAP.

To identify up- and down-regulated GO at each selected time point, differentially expressed genes of all pairwise comparisons were subjected to GO enrichment analysis (Figure S3). At the transition from UNP to 12 HAP, ‘cell wall organization’, ‘cell wall modification’, ‘methylation’, ‘cell growth’, ‘developmental growth involved in morphologies’, ‘DNA replication’ were upregulated. However, ‘organ nitrogen compound catabolic processes, ‘oxidation-reduction process’, ‘lipid catabolic process’ were downregulated. At the transition from 12HAP to 48HAP, ‘Carbohydrate metabolism process’, ‘photosynthesis, dark reaction’, reductive pentose-phosphate cycle’ ‘polysaccharide metabolism (glucan)’, ‘electron transport in photosystem I’ were significantly upregulated. Downregulated GO terms were mainly ‘chitin metabolic process, ‘amino sugar metabolic processes’, ‘glucosamine catabolic process’. At the transition from 48HAP to 96HAP, ‘cell wall metabolic and biosynthetic process’, ‘xylan biosynthetic process’, ‘negative regulator of peptidase and hydrolase activity’ was significantly upregulated. The overrepresentation of ‘microtubule-based movement’, ‘cell division’ ‘mitotic cell cycle’, ‘cytokinesis’ was enriched in the downregulated GO group (Figure S3).

Tryptophan-dependent auxin biosynthesis pathway after pollination

In higher plants, auxin is biosynthesized from the tryptophan (Trp) by the indole-3-pyruvic acid (IPA) pathway [17]. It’s a two-step process that involves the amino group removal from Trp forming indole-3-pyruvate (IPA), catalyzed by TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) family and then IPA decarboxylation by YUC flavin mono-oxygenase enzymes (YUC), forming IAA. In this study, 1 TAA (Spo25321) and two YUC (Spo11200, Spo24134) transcripts were found to be upregulated at UNP vs 12HAP (Fig. 2a, Table S2).

KEGG analysis emphasized the differential gene expression of genes involved in a Trp-dependent auxin biosynthesis and b auxin signal transduction pathway

Phytohormone signal transduction insight reveals enrichment in auxin signaling pathway

KEGG enrichment analysis revealed regulation in phytohormone signal transduction after pollination. A deep insight into this pathway revealed the clearly defined enrichment in auxin transport and signaling pathway (Fig. 2b, Table S2). Auxin is unique among other plant hormones for having polar transport due to its weak acidic nature and its intercellular movement is accomplished by auxin influx AUXIN1/LIKE-AUX1 (AUX/LAX) and auxin efflux carriers PIN-FORMED (PIN) proteins. Three AUX1 transcripts expression found to be accumulated after pollination with two transcripts at UNP vs 12HAP and one at 12 vs 48 and 48 vs 96HAP. Besides that, 2 PIN transcripts downregulated at 12 vs 48 h after pollination suggest further auxin accumulation. Two AUX/IAA (auxin response factors repressors) genes showed non-significant change in expression pattern at UNP vs 12 HAP, which promotes the transcription of ARF (AUXIN RESPONSIVE FACTORS) at 12 HAP. ARF mediated early small auxin up-regulated RNA genes (14 SAUR) were differentially regulated after pollination with upregulation of 12 SAUR genes at UNP vs 12HAP, however, only a few genes were upregulated at 12 vs 48HAP and 48 vs 96HAP. Decrease expression of some Gretchen Hagen 3 (GH3) transcripts such as GH 3.1 and GH3.6 involved in auxin conjugation, suggested that there would also be some active auxin accumulation at later time points. Expression data suggest that significant auxin signaling is induced at 12 HAP to regulate growth responses at later time points.

Cell expansion and cell division are altered after pollination

Surveying the auxin signal transduction pathway clearly showed a large portion of DEG’s after pollination. Auxin is a major regulator of plant development and growth. Many aspects of these processes involved multiple auxin exerted controls on cell wall expansion and cell division. Further, a significant expansion in cell size was observed after pollination as sepal grows (Fig. 6A). This led us to raise a question about (1) cell expansion, modification, and division gene expression increase after pollination. To address this, we examined expression pattern of genes involved in these pathways. Cell expansion requires cell wall loosening that is primarily regulated by endotransglycosylase/hydrolases (XTHs) and expansins (EXP). Sixteen putative members of the expansin family were differentially expressed in the pollinated sepals. All these genes were up-regulated at UNP vs 12 h after pollination except for 1 gene that has up-regulation at 48 vs 96HAP. Eight genes among 16 expansin genes were downregulated through the 12 to 48HAP and 48 to 96HAP transitions. A total of 8 genes were annotated as XTHs were differentially expressed after pollination, and these genes were up-regulated at 48 vs 96HAP with 4 genes also up-regulated at UNP vs 12HAP. In addition, the modulation of several genes involved in cell wall modification and homogalacturonan breakdown was observed. Twenty-three pectinesterase genes were regulated after pollination. Seventeen of these genes were up-regulated at UNP vs 12HAP and 6 genes at 12 vs 48HAP. Downregulation of 9 and 11 genes was observed at 12 vs 48 and 48 vs 96 h after pollination, respectively. This may indicate that the pectin polymers are broken down rapidly for recycling after pollination. Enrichment of most of the cell wall loosening and pectinesterases may suggest that cell enlargement signals start rapidly right after pollination at 12H and continue at later time points (Table 1).

We then surveyed key genes involved in cell division that encompasses the sequence of events [18]. The initiation of active replication (S-phase) requires the assembly of proteins including replication factors RFA, minichromosome maintenance protein complex (MCM), DNA polymerases, proliferating cell nuclear antigen (PCNA), and other factors. Five MCM genes (MCM 2, 3, 4,6,7), two DNA polymerase α primase complex genes (PRI1and POLA2), two PCNA genes, and three replication factor (RPA1, RPA2, and RFC3) were found to be up-regulated at UNP vs 12HAP, while no regulation was observed through12hap to 48hap and 48hap to 96hap transitions (Fig. 3b, Table S2). Cyclins, Cdks, and APC/C involved in cell cycle phase transitions were found to be regulated after pollination. Eleven genes annotated as cyclin, key cell cycle regulators triggering G1 to S and G2 to M transitions [19] were differentially expressed after pollination. Five genes showed high homology to cyclin-A, 5 genes with cyclin B, and 1 gene with cyclin D was upregulated at UNP vs 12HAP. Among these, 2 cyclin A and 4 cyclin B genes were downregulated through 48HAP to 96HAP transition. None of these genes were found to be regulated at 12HAP vs 48HAP. Cyclins regulate the cell cycle events by partnering with an enzyme family called cyclin-dependent kinases (Cdks). Two CDKB genes (CDKB1 and 2) were upregulated only at UNP vs 12HAP while downregulated at 48 vs 96HAP. The Anaphase-promoting complex (APC/C), another cell cycle regulator causes protein degradation that holds sister chromatids and allowing them to move to opposite poles of the cell during anaphase. They also cause M cyclins degradation, allowing the new daughter cells to enter G1 by pushing the cell out of mitosis. Two APC gene homologous to APC8 and 10 were upregulated at UNP vs 12HAP and not regulated through the 12HAP to 48HAP and 48HAP to 96HAP transition (Fig. 3a). Enrichment of replication machinery genes and cell cycle regulators genes indicate that the cell number of sepal organ start increasing after pollination.

Differential expression of gene involved in cell-cycle. a Cell cycle checkpoints. b DNA replication

Cell wall metabolism is altered after pollination

A possible direct or indirect role of auxin in cell wall polysaccharides synthesis and the over-representation of cell wall-related genes in GO enrichment analysis led us to study these genes in detail (Fig. 4, Table S2). Cell wall is composed of cellulose, hemicellulose, pectin, and lignin, which cross-link and interact to form a complex and rigid network. Cellulose is the most abundant biopolymer in plant cell wall and composed of UDP-glucose that is catalyzed by cellulose synthase (CS). Two CESA genes homologous to Arabidopsis CESA4 and 8, essential components of the CESA complex in SCWs, were found to be upregulated at 48 vs 96HAP. COBRA genes encode GPI-anchored proteins involved in crystalline cellulose assembly during cell wall formation. Three COBRA-like genes were predominantly expressed at 48 vs 96HAP, with a similar expression pattern to CESA genes indicate that they might cooperatively involve in cellulose assembly and synthesis in cell wall. Hemicellulose is the second important cell wall component, catalyzed by cellulose synthase-like genes and glycosyl-transferases (GT). Four CSL genes were found to be differentially expressed after pollination. Among them, 3 genes were upregulated at UNP vs 12HAP and 48 vs 96HAP. In addition, five members of GT genes were preferentially expressed at 48 vs 96HAP. Lignin is composed of amorphous polymers monolignols, which are biosynthesized by phenylpropanoid pathway. In total, 12 genes involved in lignin biosynthesis pathway were significantly expressed after pollination. As expected, several lignin biosynthesis transcripts including one hydroxycinnamoyltransferase (HCT), three caffeoyl CoA 3-O-methyltransferase (CCoAOMT), two cinnamyl alcohol dehydrogenase (CAD), one cinnamoyl-CoA reductase (CCR), and five catechol-O-methyltransferase (COMT) were differentially expressed after pollination. Finally, the monolignols are polymerized by peroxidases and laccases, then transported to the cell wall. Eight laccase (LAC) and 17 peroxidase genes were differentially expressed after pollination with most genes were dramatically upregulated at 48 vs 96HAP. Among numerous enzymes, glycosyl hydrolases (GHs) are associated with cell wall polysaccharides degradation and remodeling. Nine GHs genes were found to be regulated after pollination. Among them, 8 genes were upregulated at 48 vs 96HAP, 3 at 12 vs 48HAP, and 2 at 12 vs 48HAP. The expression patterns of these genes were consistent with the substantial lignification and cell wall biosynthesis genes at 96 h after pollination.

Differential expression of gene involved in cell wall metabolism

Photosynthesis and chlorophyll contents are altered in pollinated flower sepal

Survey of cell wall metabolism clearly indicates a large portion of DEGs belongs to cellulose, hemicellulose, monolignol polymerization, cell wall loosening, and degradation after pollination. UDP-glucose is the major building block of cell wall and the intermediate in sucrose biosynthesis pathway. This led us to look at the expression pattern of Calvin cycle genes (Fig. 5a, Table S2). We found dynamic changes in gene expression pattern through the 12 to 48HAP transition in all three phases of the Calvin cycle (fixation, reduction, and regeneration) regulated by specific enzymes. Two rubisco small subunit genes, involved in carbon fixation, one phosphoglycerate kinase, and one glyceraldehyde phosphate dehydrogenase (GAPDH) having important roles in photosynthetic carbon reduction found to be upregulated through 12HAP to 48HAP transitions. However, three genes (aldolases and transkeletoses) involved in regeneration of Ribulose 1,5-bisphosphate step that limits photosynthesis found to be upregulated at 48 vs 96HAP. Since sepal enhanced the Calvin cycle after pollination, it was considered that the light-dependent reaction of photosynthesis also intensifies simultaneously to provide metabolic energy (Fig. 5b). The up-regulation of light-harvesting center proteins (4 genes of photosystem II and 2 genes of photosystem I) together with other photosynthesis components such as the electron transporter ferredoxin and ATP-synthase suggested that the photosynthesis apparatus might be attenuated notably at 12 vs 48HAP. Next, we examined whether there is conversion of sepal photosynthate to hexose after pollination. Sucrose synthase (SuSy) reversible convert sucrose to UDP-glucose which is the substrate for biosynthesis of cellulose and the other nucleotide-sugar precursors required for hemicellulose and pectin. In this study, only one Susy was up-regulated at 12 vs 48 and 48 vs 96HAP. As chlorophyll is widely considered the direct regulator of photosynthetic capacity in plant leaf (Singsaas et al., 2004), we then measured the sepal chlorophyll contents at each time point after pollination (Fig. 6B). The results indicated that total chlorophyll contents increase at 12, 48 HAP when sepal growth just starts, and then slight decrease at 96HAP, but still more than unpolllinated samples. A significant decrease in these contents was observed after 10 days of pollination when sepal has been grown enough to suggest that photosynthetic activity enhances right after pollination to provide precursor for growth and eventually drops when significant growth occurs.

Display of gene expression of genes. a Calvin cycle. b Photosynthesis light dependent reaction

A. (i) Unpollinated flower sepal cells (ii) Sepal cells right after pollination (iii & iv). Cell expansion can be observed when considerable sepal is developed after pollination. B. Chlorophyll contents at different time points after pollination

RNA-seq data validation by qRT-PCR

To validate expression pattern of genes identified by RNA-seq data, 14 genes were randomly selected and examined by qRT-PCR. Two of the auxin biosynthesis genes (Spo25321, Spo24134), three genes from auxin signal transduction pathway (Spo01712, Spo23966, Spo13608), three cell cycle genes (Spo08502, Spo10811, Spo02886) characterized by higher expression at 12HAP compared to UNP. Quantitative PCR analysis confirmed differences in transcript abundance between cell wall biogenesis genes (Spo09254, Spo04584) and SAUR (Spo22272) at 48 vs 96HAP, much higher than12 vs 48 HAP and UNP vs 12HAP. Two cell expansion genes (Spo16879, Spo06997) and AUX1 (Spo10854) genes also showed similar expression patterns in RNA-seq and qPCR. Expression patterns of all selected genes were confirmed to be consistent with the RNA-seq data (Fig. 7).

qPCR analysis for expression confirmation, Blue bars represents the relative expression in qPCR, and orange line represents the log2 (FC) values in transcriptome for corresponding genes

Auxin acts as a signal and triggers autonomous spinach sepal development

RNA-seq analysis underlined that sepal development initiating after pollination may drive by cell expansion, cell division, and cell wall remodeling. Given that auxin signaling is active in the developing sepal after pollination, and this hormone is known for having a role in cell growth and expansion, we tested whether exogenous application of auxin provides signal to drive sepal development. We treated unpollinated spinach flower with synthetic 2,4-D and natural IAA auxin analog at 50, 200, 500, 1000uM concentration, and investigated autonomous sepal development after 10 days (Fig. 8a). We measured the size of the 15–20 sepals to estimate the average growth. At 50, 200, and 500uM, unpollinated flower sepal develop almost equally, and their growth was comparable with that of pollinated flower sepal but at 1000uM sepal wilted and did not grow at all for both synthetic with a slight increase at natural auxin analog, which indicates that over-accumulation of auxin hormones in the integuments (sepal) of spinach flower would trigger sepal abscission responses. To further confirm the effect of auxin, we treated spinach flower with NPA at 0.01, 0.1, 1 mM concentration at12HAP and investigated autonomous sepal development after 10 days (Fig. 8b). At 0.01 mM, flower sepals normally develop as mock-treated ones slightly smaller in size, but at 0.1 and 1 mM sepal development varies significantly, At 0.1 mM sepals grow a bit but smaller than 0.01 mM and mock-treated ones, at 1 mM sepal did not grow at all. We also observe the difference in seed size in pollinated flowers after NPA treatment at maturation. At 0.01 mM concentration, normal seeds as mock-treated ones, while aborted seeds of small size at 0.1 and 1 mM NPA concentration were developed. We further check the germination percentage of the seeds in each NPA treatment individually (Fig. 8c). A hundred seeds of each treatment were observed for germination and the germination percentage was calculated. In mock-treated ones, the germination percentage was about 97%. In 0.01 mM, about same germination percentage was observed as in mock-treated seeds as their sepal size was also similar, while in 0.1 and 1 mM NPA treatment, no seed germinated and consistent with their sepal size which also not developed at all. This might suggest that auxin transport through an interactive pathway may drive ovule and sepal development in spinach after pollination as sepal grows with the encased developing seeds. The quantitative data of sepal size in each treatment is illustrated in Fig. 8d.

Developmental changes observed after 10-days of auxin and auxin transport inhibitor treatment. a IAA treated unpollinated flower’s sepals at mock-, 50, 200, 500, and 1000 μM. b 2,4D treated unpollinated flower’s sepals at mock-, 50, 200, 500, and 1000 μM. c NPA-treated pollinated flower’s sepals after 12HAP at mock-, 0.01, 0.1, and 1 mM concentration gradient in female spinach variety “Cornel-9”. d Quantitative sepal development (cm) under hormone treatments at different concentrations. e Germination percentage of seeds obtained from NPA-treated pollinated flowers at maturity

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4.1 Advantages of gamma-ray imaging in intact plants

Aspects of metal uptake and homeostasis in plants may be understood well at the molecular level, but understanding of the whole-plant dynamics has lagged behind due to the limitations of traditional experimental approaches and imaging systems. Radiolabeled molecules are widely used to measure the transport dynamics in biological systems. In experiments with whole plants and radiolabeled molecules, the biodistribution of the radiolabel is most typically analyzed by plant dissection and counting in a well counter, and for β-emitters, by ashing the plant biomass and counting the radiolabel with a liquid scintillation counter. Several whole plant positron emission tomography (PET) imaging systems have been developed using 11 C (Jahnke et al., 2009 Kawachi et al., 2011 Weisenberger et al., 2009 ), other groups have developed large scanners for β-imaging using 32 P (Kanno et al., 2007 ), and autoradiography has been used to image radioisotope distribution of whole plants (Page & Feller, 2005 ). A scanner (Kawachi et al., 2011 ) has been used to perform PET imaging of 65 Zn and 107 Cd in rice plants (Fontanili et al., 2016 Suzui, Yin, Ishii, Sekimoto, & Kawachi, 2017 ). Each of these nuclear imaging methods has drawbacks: for β-imaging, the range of β-particles can be too short to escape the plant for PET, radioisotope lifetimes are short and the range of the positron can be too long for a thin, low density plant, limiting the yield of annihilation gamma-rays and autoradiography is invasive and can require exposure times of weeks or months. Therefore, UCD-SPI for nuclear imaging of gamma-ray emitters in plants has significant opportunity to contribute in a new way to transport studies. Indeed, the UCD-SPI system has extremely high sensitivity (

10%) for a single-photon imaging system, orders of magnitude higher than is found for SPECT systems used in medical imaging (often

0.01%). This is extremely useful for imaging over long periods of time with high energy gamma rays, as it allows for imaging very low levels of radiotracer and thereby eases the practical issues of radiation safety and shielding and waste generation.

In particular, the uniquely high sensitivity of the system means that very small amounts (nCi) of radioisotopes may be imaged and followed over time. The system used here had the advantage that one entire time course can be recorded on a single plant. In time courses involving destructive sampling or imaging, data for the different time points are from distinct plant individuals, which generates substantial noise. This is particularly disruptive in experiments with non-model plants such as A. halleri, which show substantially larger variation in plant architecture between independently grown plants even of an identical genotype.

Common PET isotopes of interest for plant studies include several found in organic compounds: 11 C (T1/2 = 20 min), 13 N (T1/2 = 10 min), and 18 F (T1/2 = 109 min). These atoms can be substituted into amino acids or sugars in plants to follow the natural in situ processes (Cherry, Sorenson, & Phelps, 2003 ). However, due to the short lifetimes of PET radioisotopes, only short biological processes, such as photosynthesis, may be imaged. In contrast, single gamma-ray emitting radiotracers used in single photon emission computed tomography (SPECT) are typically metals and do not easily label organic molecules. However, many trace element metals are essential to a plant's survival (Clemens, 2001 Williams, Pittman, & Hall, 2000 ). An active area of plant research is studying hyperaccumulation of metals in plants using a radioisotope of that metal commonly studied metals include: Cd, Zn, Mn, Co, and Ni (Krämer, 2010 Page & Feller, 2005 ). Other potential applications for SPECT imaging include: studying plant ion transport in xylem (Macklon, 1970 Ueno et al., 2008 ), studying metabolic processes such as tracers for phloem transport (Omid, Malter, Peleg, & Wolf, 2008 ), and studying signaling by labeled exogenous peptides or proteins (Santner & Estelle, 2009 ). One further advantage of imaging systems based on gamma-ray detection is the possibility of detecting the interactions of multiple radioisotopes simultaneously as the gamma-rays that they emit have distinct energies that can be distinguished from each other by the detector. For example, simultaneous imaging of 65 Zn (gamma-ray 1,116 keV) and 109 Cd (22 keV) would enable teasing apart the competition dynamics in their uptake.

However, there is an inherent tradeoff in increased sensitivity of the UCD-SPI system with spatial resolution. Spatial resolution at the mm-scale could be obtained using a collimator to better define the spatial origin of detected gamma rays, but this would lead to a greatly reduced event rate in the system. For the high-energy gamma rays of 65 Zn, collimation is a particular challenge. Given the hours-long time scale of the transport studied here, it is possible that the choice of a collimator could have provided improved event positioning while preserving a usable event rate. A possible hybrid approach could have included using an insertable/removable collimator to acquire an alternating combination of two types of images: high sensitivity-low spatial resolution without the collimator and low sensitivity-higher spatial resolution with a collimator. However, the use of a collimator for this system is unexplored thus far.

4.2 Zn transport dynamics in A. halleri and A. thaliana

Zn uptake into the symplast in the outer root layers and loading into the apoplastic xylem stream are well understood on molecular level (Moreira, Moraes, & dos Reis, 2018 ). However, the dynamics of symplastic movement and patterning of the radial transport have thus far only been modeled to elucidate the timescales of these events (Claus et al., 2013 ). After xylem loading, the mass flow-mediated movement of Zn into the shoot inside the xylem is expected to occur within 30 min in Arabidopsis, as previously shown for water (Park et al., 2014 ) and Cd in xylem sap (Ueno et al., 2008 ). From previous SPECT imaging, we have shown that a pulse of radiolabelled pertechnetate ( 99m TcO4 − ) moving in the xylem stream reaches the shoot apical meristem of a 2 week old sunflower already in 5 min (Walker et al., 2015 ).

The rate-limiting step for root-to-shoot translocation of Zn was proposed to be xylem loading involving HMA4 transporters in both A. halleri and A. thaliana (Hanikenne et al., 2008 Sinclair, Sherson, Jarvis, Camakaris, & Cobbett, 2007 ). The dynamics of root-to-shoot Zn flux, however, have so far remained unclear in different species and transgenic lines. Estimates of Zn translocation rates from root to shoot were first obtained by spectroscopy methods of ashed shoot tissues. Early work with metal hyperaccumulator Noccaea caerulescens suggested that the speed of root-to-shoot Zn transport was between 20 and 60 hr (Lasat, Baker, & Kochian, 1996 Lombi, Zhao, McGrath, Young, & Sacchi, 2001 ). Recently, positron imaging of Zn uptake estimated the time for Zn root-to-panicle transport in dwarfed mature rice to be 5.3 hr (Suzui et al., 2017 ). Here, we have produced the first Zn root-to-shoot imaging data for A. halleri using UCD-SPI. Zn accumulates within the shoot of A. halleri, consistent with its ability to hyperaccumulate Zn, different from the HMA4 RNAi line. The speed of Zn transport into the shoot in our data as observed with the smoothed standard error show clear shoot accumulation within 5–7 hr, respectively (Figure 2c). These results are in line with previous reports for rice (Suzui et al., 2017 ). This contrasts strongly with the faster speed of the other xylem-transported compounds, such as water in A. thaliana (Park et al., 2014 ), Cd in A. halleri (Ueno et al., 2008 ) and pertechnetate in sunflower (Walker et al., 2015 ), all measured to reach the shoot in 30 min. It should be noted, however, that the experiments demonstrating water transport (Park et al., 2014 ) and Cd transport (Ueno et al., 2008 ) were carried out using decapitated stems and are thus destructive in nature, but also far more sensitive to small quantities than the method used here. The slower speed of Zn transport indicates that Zn loading into the xylem by HMA4 is slow and under tight control even in the metal hyperaccumulator A. halleri. Modelling the radial transport of Zn uptake has indeed indicated that HMA concentration is one of the key determinants of the uptake dynamics (Claus et al., 2013 ).

The HMA4 transporter pumps Zn 2+ from the root symplasm into the apoplastic xylem sap of A. thaliana (Verret et al., 2004 ). Strongly elevated expression of A. halleri HMA4 was suggested to be responsible for the increased in root-to-shoot translocation of Zn in A. halleri relative to A. thaliana (Hanikenne et al., 2008 ). This conclusion was drawn based on the quantification of shoot Zn concentrations after long-term growth in HMA4-RNAi lines and wild-type A. halleri and in A. thaliana Col-0 (Hanikenne et al., 2008 ). In the same experiment, root Zn concentration was elevated in some A. halleri HMA4 RNAi lines relative to A. halleri wild-type plants and even relative to A. thaliana (Hanikenne et al., 2008 ).

HMA4 is critical to the ability of A. halleri to hyperaccumulate Zn. We tested the functional role of HMA4 for A. halleri Zn translocation from root to shoot by imaging the Zn uptake dynamics of A. halleri HMA4-RNAi line relative to A. halleri. We found that the Zn signal in the shoot of HMA4-RNAi line did not increase over our 40-hr imaging period, but conversely, we saw a continuous decrease in shoot Zn signal with significant differences observable at 3 hr (Figure 2c). The lack of an increase in shoot Zn confirms that Zn loading into the xylem is abolished in the HMA4-RNAi plants (Hanikenne et al., 2008 ). The continuous decrease in the Zn signal in the shoot ROI seems to reflect bleeding of the strong early Zn signal from the root ROI into the shoot ROI. The dissipating signal through the A. halleri HMA4-RNAi time course could be due to apoplastic 65 Zn adsorbed to the cell walls of outer root layers during the 65 Zn pulse (Lasat et al., 1996 ) and not removed by the triple rinsing with Hoagland solution. This cell wall-adsorbed 65 Zn would be desorbed into the growth medium during the imaging period by diffusion. The influx of Zn into the root symplasm is very tightly and rapidly regulated in Zn-concentration dependent fashion (Claus et al., 2013 van de Mortel et al., 2006 Talke et al., 2006 ). Without the loading of Zn into the xylem, Zn builds up in the root symplasm. In the case of A. halleri HMA4-RNAi, the symplasm could be saturated with Zn at 3 hr after the resupply, leading to prevention of further uptake of the cell wall-adsorbed 65 Zn and thus higher Zn desorption than Zn uptake into the symplasm.

Finally, we compared the dynamics of Zn movement in the Zn hyperaccumulator A. halleri with those in the related species A. thaliana, a non-metal hyperaccumulator. Based on previous studies comparing Zn-deficient to Zn-sufficient plants of A. thaliana and/or A. halleri, the Zn concentrations in our hydroponic solutions can be estimated to result in moderate Zn deficiency (Talke et al., 2006 Sinclair et al., 2018 ). The net concentration of 65 Zn in the resupply media over the 24 hr period showed a net decrease, suggesting that Zn was taken up into the shoot, although these levels are variable. We found that Zn resupply after Zn deprivation in A. thaliana did not lead to detectable uptake or change of Zn in the shoot or the root ROI. It is possible that the small size and flat rosette growth habit of A. thaliana affected our ability to detect Zn dynamics. Also, low abundance of HMA4 transporters in A. thaliana roots may lead to much slower dynamics that we were unable to capture. In the absence of quantification of 65 Zn levels in the shoot, it is possible, although unlikely, that Zn was not translocated in A. thaliana.

The heavy metal imaging study presented here is of interest for phytoremediation applications (Kärenlampi et al., 2000 Krämer, 2010 Robinson et al., 1998 Salt, Smith, & Raskin, 1998 Sarma, 2011 ). Although most plants prevent the accumulation of heavy metals so as to avert toxicity, metal hyperaccumulators selectively extract high concentrations of metals from the soil into their shoots without incurring symptoms of toxicity (Baker & Brooks, 1989 Frérot et al., 2010 ). By using the heavy metal radiolabel 65 Zn and the UCD-SPI imaging system, we gained a more detailed spatiotemporal understanding of the dynamics of metal movement into plants, which may be a path toward the use and understanding of metal hyperaccumulating plants for such advantageous applications.


The closing line of The Nervous System of Plants reads: “No structure corresponding to the nerve-ganglion of an animal has, indeed, been discovered in the pulvinus of Mimosa pudica, but it is not impossible that the physiological facts may one day receive histological verification.” (Bose, 1926 , p. 218).

Although Bose failed to find an analogous equivalent, the “glomerulus” composed of a complex stack of interconnected phloem bundles and several millimetre in length suggests one might well exist (Behnke, 1990 ). This phytoneurological system is highly cross-linked. Figure 1 (fig. 54 from Bose, 1926 ) shows the vascular system of Papaya to consist of vascular elements cross-linked extremely frequently by numerous, irregularly distributed and tangential connections. A network of excitable phloem cells is clearly present. “How reticulated they (the vascular bundles) may often be, even in the trunk of a tree, is shown in the photograph of the distribution of vascular bundles in the main stem of Papaya …. This network of which only a small portion is seen in the photograph girdles the stem throughout its whole length and in this particular case, there were as many as twenty such layers one within the other” (Bose, 1926 , p. 121).

In very young plants, such as Helianthus seedlings, phloem anastomoses (cross links), up to 7,000/stem internode in number, have been reported. How common this cross linking might be remains unknown (Aloni & Barnett, 1996 Aloni & Sachs, 1973 ). It is speculated that auxin might be responsible for their formation, and that they might have a function in xylem regeneration. Computer-assisted tomography has been used to identify a complex network of xylem vessels (Brodersen et al., 2011 ). However, xylem does not differentiate in the absence of phloem, although the converse is not true (Roberts, Gahan, & Aloni, 1988 , p. 47). The observed vessel network probably indicates the phloem network too.

In more mature stems and trunks, with the appearance of additional secondary and supernumerary cambia, and other features of secondary growth, plant vascular architecture becomes extremely complex. Tangential connections and anastomoses between numerous bundles become very frequent as do radial connections between different stem layers (Carlquist, 1975 Dobbins, 1971 Horak, 1981 Wheat, 1977 Zamski, 1979 ). These anastomoses do not occur simultaneously in the xylem and phloem but construct a “complex net-like structure” already observed in some related 20 families of plants (Zamski, 1979 ). The complexity of the excitable phloem network is nothing like the simple structures of vascular tissue presented in text books that are usually limited to seedlings. Woody tissues, often xylem, are sometimes penetrated by interxylary phloem. Starch is deposited in the xylem that is then mobilized on a seasonal basis.

5.1 Importance in establishing the presence of a network.

Even very simple networks of some five interconnected nerve cells using all-or-none action potentials exhibit a capability for memory, error correction, time sequence retention, and a natural capacity for solving optimisation problems (Hopfield, 1982 Hopfield & Tank, 1986 McCulloch & Pitts, 1943 ). Some of these capabilities are present in plants although they are not specifically identified with the phloem system (Trewavas, 2017 ). Thus, knowing the complexity of this phloem based network might improve understanding of these behavioural properties of plants.

Is this network and its behaviour sufficiently complex in behaviour and memory to be analogous to mental states? Again, we cannot comment until the network complexity is better understood, and the frequency and particular qualities of the cross linkages investigated.

Different applications of chlorophyll fluorescence

Relationship to CO2 assimilation

In whole-leaf studies it is natural to extend the interpretation of chlorophyll fluorescence data to analyse its impact on photosynthetic rates of CO2 assimilation and, by inference, productivity of the plant or system in question.

Under certain controlled circumstances, the Fq′/Fm′ measured by fluorescence is accurately correlated with rates of CO2 assimilation which has added extra interest to extend the possibilities of this technique and has led to advances in our understanding of photosynthetic regulation ( Genty et al., 1989, 1990 Cornic, 1994). This relationship makes intuitive sense because the products of linear electron transport, ATP and NADPH, are used directly in photosynthetic carbon assimilation in known ratios. In C3 plants, this close correlation can be observed best when photorespiration is inhibited by lowering the partial pressure of oxygen to 2%. Why? The electron requirement for assimilation of one CO2 molecule in leaves where photorespiration is inhibited is four. This number will rise as the proportion of carbon flux through the photorespiratory pathway rises, for example as happens during stomatal closure ( Wingler et al., 1999 Flexas et al., 2002). Therefore, whenever the electron requirement is altered, the relationship between Fq′/Fm′ and CO2 assimilation rate is also altered. Unfortunately, for field-based measurements, the number of factors that can cause this is high: stomatal closure (as mentioned below), temperature (this changes the relative rates of carboxylation and oxygenation in the leaf), abiotic stress (this results in an increased activity of alternative electron sinks such as the Mehler reaction and cyclic electron transport), leaf development, and shading. A common example given of this disconnect between Fq′/Fm′ and the CO2 assimilation rate is that of species in environments undergoing multiple stresses ( Cheesman, 1991). In conclusion, this means that linearity is difficult to achieve unless tightly controlled conditions are used and care is taken when comparing samples in which the ratio of allocation between CO2 assimilation and other processes is known not to have changed. The relationship in C4 plants is much more easily achieved due to the suppression of photorespiration by the CO2-concentrating mechanism. Another point of error is the one mentioned above regarding the accurate measurement of ETR.

Imaging of chlorophyll fluorescence

Imaging of chlorophyll fluorescence is becoming increasingly popular as a screening ( Barbagalo et al., 2003) and diagnostic tool ( Baker, 2008), due mostly to the development of instrumentation ( Oxborough, 2004), with many commercial instruments available through companies such as Photon Systems Instruments (Brno, Czech Republic) Walz (Effeltrich, Germany), and Technologica Ltd (Essex, UK), amongst others. Chlorophyll fluorescence imaging has also recently been incorporated into many phenotyping platforms for high-throughout phenotypic analysis. There are several advantages to imaging, and below we have explored some of these along with some examples of uses.


Chlorophyll fluorescence imaging allows multiple plants to be monitored at the same time under identical conditions, providing an ideal screening platform. Chlorophyll fluorescence directly relates to the rate of energy flow via the electron transport chain and therefore any perturbation that impacts on plant metabolism (e.g. pathogen infection) will impact on fluorescence parameters even if not directly linked to photosynthesis ( Barbagallo et al., 2003). Examples of screening multiple plants include early detection of herbicide application ( Barbagallo et al., 2003 Jin et al., 2011), nutrient deficiency ( Mauromicale et al., 2006), drought stress ( Rahbarian et al., 2011), identification of CO2-sensitive photorespiratory Arabidopsis thaliana mutants ( Badger et al., 2009), and plants with improved photosynthesis and crop yield ( Baker and Rosenqvist, 2004 Chaerle et al., 2007 Baker, 2008 Harbinson et al., 2012).

Spatial and temporal heterogeneity

An advantage of any imaging technique is the detailed spatial representation of the measured parameter, allowing the assessment of sample heterogeneity. Monitoring changes within such images provides additional temporal assessment of the measured parameter. Examples of spatial heterogeneity highlight the value and appeal of imaging, by drawing attention to the fact that ‘detection of symptoms’, or assessment of ‘reductions in a specific fluorescence parameter’ may well have been missed if a traditional fibre optic approach had been utilized, which is only capable of measuring a small area/proportion of the leaf. There are numerous examples of chlorophyll fluorescence imaging for detecting within-leaf or plant treatment effects, including the assessment of freezing tolerance and cold acclimation in Arabidopsis (e.g. Ehlert and Hincha, 2008), insect herbivory (e.g. Tang et al., 2006), leaf fungal infection (e.g. Scholes and Rolfe, 2009 McElrone et al., 2010), and the impact of ozone damage ( Leipner et al., 2001 Aldea et al., 2006).

Degrees of resolution

Depending on instrumentation, images of chlorophyll fluorescence can be obtained at a range of resolutions. High-resolution images of stomatal guard cells have been used to determine efficiency and quenching parameters from individual chloroplasts ( Baker et al., 2001 Fig. 3). The spread of disease and host–pathogen interactions has been monitored both spatially and temporarily at the leaf level using high-resolution images of NPQ and Fq′/Fm′ (Scholes and Rolfe, 2008), while Aldea et al. (2006) illustrated fine-grain heterogeneity in mapping the spatial patterns of the production of reactive oxygen species (ROS) on PSII quantum efficiency following viral infection or ozone damage. A high-resolution microscope imaging system was used to show the impact of water stress and CO2 concentration on guard cell photosynthetic efficiency in a range of different plant types by Lawson et al. (2003). A similar system has been used to detect the impact of heavy metals such as cadmium on the aquatic plant Lemna ( Fig. 3) Three-dimensional chlorophyll fluorescence imaging has also been used in the detection of herbicide effects on a whole plant ( Eguchi et al., 2008).

(a) Images of steady-state fluorescence (F′) and (b) photosynthetic efficiency of PSII photochemistry (Fq′/Fm′) from 10cm 2 leaf area enclosed in a chamber during simultaneous gas exchange measurements demonstrating the effect of patchy stomatal closure. Images were captured 5min after dropping the humidity rapidly from 80% to 20%. All other chamber conditions were maintained at 200 μmol m –2 s –1 PPFD, 400 mmol mol –1 [CO2], 2% [O2], and a temperature of 24 °C. (c) An example of chlorophyll fluorescence as a screening technique showing the detection of the effects of herbicide treatment on Fv/Fm of 1-week-old Arabidopsis plants growing on a 96-well plate (unpublished image of Technologica Ltd, with permission). (d) An Fq′/Fm′ image showing the heterogeneity of two Arabidopsis plants after 10min acclimation to 300 μmol m –2 s –1 PPFD following induction of heterogeneity with 1h at a high light intensity of 1000 μmol m –2 s –1 . (e) An image of Fv/Fm of the epidermis of an intact Tradescantia leaf showing the guard cell chloroplasts following the methods of Oxborough and Baker (1997b) taken with a high resolution microscope chlorophyll fluorescence imaging system (unpublished image of McAusland and Lawson). (f) Use of the chlorophyll fluorescence parameter Fq′/Fm′ to screen transgenic plants that have reduced levels of SBPase. The plant in the upper right and lower left have much greater reductions in SBPase than the other two plants. (g) Images of Fv/Fm taken after 20min dark adaption from three different varieties of wheat plants grown in the field, with different susceptibilities to rust. The middle images show a variety that is more susceptible to rust (unpublished image of Lawson and Driever). (h) A cut elder leaf illustrates the decrease in photosynthetic efficiency (Fq/Fm′) with DCMU uptake via the petiole during transpiration. (i) High-resolution image of the effect of Cd on photosynthetic efficiency in the aquatic plant Lemna. The image was captured after stabilizing to 200 μmol m –2 s –1 light after having been exposed to Cd for 1 d. The image on the left represent steady-state fluorescence (F′) while that on the right is a false-colour image of Fq′/Fm′. (Unless stated all images are unpublished data of TL.)

(a) Images of steady-state fluorescence (F′) and (b) photosynthetic efficiency of PSII photochemistry (Fq′/Fm′) from 10cm 2 leaf area enclosed in a chamber during simultaneous gas exchange measurements demonstrating the effect of patchy stomatal closure. Images were captured 5min after dropping the humidity rapidly from 80% to 20%. All other chamber conditions were maintained at 200 μmol m –2 s –1 PPFD, 400 mmol mol –1 [CO2], 2% [O2], and a temperature of 24 °C. (c) An example of chlorophyll fluorescence as a screening technique showing the detection of the effects of herbicide treatment on Fv/Fm of 1-week-old Arabidopsis plants growing on a 96-well plate (unpublished image of Technologica Ltd, with permission). (d) An Fq′/Fm′ image showing the heterogeneity of two Arabidopsis plants after 10min acclimation to 300 μmol m –2 s –1 PPFD following induction of heterogeneity with 1h at a high light intensity of 1000 μmol m –2 s –1 . (e) An image of Fv/Fm of the epidermis of an intact Tradescantia leaf showing the guard cell chloroplasts following the methods of Oxborough and Baker (1997b) taken with a high resolution microscope chlorophyll fluorescence imaging system (unpublished image of McAusland and Lawson). (f) Use of the chlorophyll fluorescence parameter Fq′/Fm′ to screen transgenic plants that have reduced levels of SBPase. The plant in the upper right and lower left have much greater reductions in SBPase than the other two plants. (g) Images of Fv/Fm taken after 20min dark adaption from three different varieties of wheat plants grown in the field, with different susceptibilities to rust. The middle images show a variety that is more susceptible to rust (unpublished image of Lawson and Driever). (h) A cut elder leaf illustrates the decrease in photosynthetic efficiency (Fq/Fm′) with DCMU uptake via the petiole during transpiration. (i) High-resolution image of the effect of Cd on photosynthetic efficiency in the aquatic plant Lemna. The image was captured after stabilizing to 200 μmol m –2 s –1 light after having been exposed to Cd for 1 d. The image on the left represent steady-state fluorescence (F′) while that on the right is a false-colour image of Fq′/Fm′. (Unless stated all images are unpublished data of TL.)

Combined with other techniques

The combination of chlorophyll fluorescence imaging with other measurement techniques and instrumentation can provide a unique research tool, that enables users to answer novel questions. For example, using chlorophyll fluorescence imaging with infra-red gas exchange (IRGA) techniques enables the user to correlate PSII photosynthetic efficiency directly to the IRGA-measured CO2 assimilation rate by eliminating photorespiration through the reduction of [O2] or increase in [CO2] within the chamber. For example, such calibration techniques have been instrumental in developing protocols and procedures to visualize patterns of CO2 diffusion in leaves ( Morison et al., 2005 Lawson and Morison, 2006), enabling the determination of gas fluxes within leaves of different species ( Morison et al., 2007), taking into account the venation patterns of the leaf ( Lawson and Morison, 2006) and the importance of fluxes on carbon gain ( Pieruschka et al., 2005, 2006 Morison and Lawson, 2007). Control of the gas environment around the samples being imaged enables users to identify specific plants traits for example, photorespiratory mutants were identified under a zero [CO2] environment, with plants maintained in an air-tight box with a layer of CO2-absorbing material on the bottom ( Badger et al., 2009). Using a high-resolution microscope chlorophyll fluorescence imaging system, Lawson et al. (2002) employed a specially designed IRGA chamber to control [CO2] and [O2] as well as humidity, to show for the first time in intact green leaves that Calvin cycle activity was the major sink for guard cell photosynthetic electron transport. Using humidity as a driver of stomatal behaviour (which does not directly affect photosynthesis), the same authors also showed that the opening and closing of stomata is not linked to guard cell photosynthetic efficiency unless the closure reduces the internal CO2 concentration to which guard cell photosynthetic electron transport responded ( Lawson et al. 2002). The combination of chlorophyll fluorescence imaging with other imaging technologies is also proving an extremely powerful tool in the development of large-scale phenotyping protocols and platforms. Although a full description of such phenotype imaging is beyond the scope of this review, the use of combined chlorophyll fluorescence imaging and thermography can supply critical information on photosynthetic rates in relation to stomatal behaviour ( Chaerle et al., 2007) and, if performed under controlled conditions with appropriate calibrations, could provide an approach for imaging intrinsic water use efficiency (iWUE) ( Lawson, 2009). Chlorophyll fluorescence imaging has also been combined with hyperspectral imaging for early detection of head blight disease in wheat ( Bauriegel et al., 2011). The advantage of this combined system enables chlorophyll degradation and the impact of different diseases to be distinguished based on changes in photosynthetic efficiency and spectral signatures such as those used in remote sensing that assess vegetation status, such as the Normalized Difference Vegetation Index (NDVI).

There are also a couple of disadvantages to imaging chlorophyll fluorescence relative to the use of a standard fibre optic system. The cost of the instruments tends to be greater than those of fibre optic fluorimeters (although inexpensive compared with buying several fluorimeters for measuring several plants simultaneously). Chlorophyll fluorescence imaging systems tend to require large banks of light-emitting diodes (LEDs) to ensure even actinic illumination and fully saturating pulses over the entire imaging area. This makes the instruments relatively large in size, and, in general, they are considered laboratory instruments and not portable systems, such as many of the hand-held devices. Having said this, several manufacturers have released field-based hand-held imaging systems (many of which are combined with IRGA instruments). For example, Walz extended its M-Series Imaging-PAM to include a MINI version for application in the field. This is a compact design, with high magnification and resolution, and can be mounted on their standard gas exchange equipment for dual measurements ( Photon Systems Instruments (, also produce a number of portable leaf chamber imaging platforms, such as ‘FluorCam’ that is designed to attach to a range of standard gas exchange chambers produced by a number of commercial manufacturers. Customized fluorescence imaging systems are also available for large-scale scanning in the field, in which the lights and camera are enclosed in a cabinet (for dark-adapted measurements) and the entire system is on wheels (, allowing it to move over crops collecting images. Large-scale measurements of chlorophyll fluorescence parameters are desirable and currently of great interest to researchers who use unmanned aerial vehicle (UAV)-based platforms for remote sensing (e.g. Zarco-Tejada et al., 2011) however, a discussion of these systems is beyond the scope of this review.

Examination of the literature illustrates many examples of uses of chlorophyll fluorescence imaging either on its own or in combination with other instruments however, due to space restrictions we have selected and illustrated only a of selection of them.

The automation and incorporation of in-built algorithms in many commercial fluorimeters has led to an increase in their use. However, as with any technique, to obtain robust and meaningful results, a suitable protocol must be designed first ( Scholes and Rolfe, 2009). The requirements for imaging are identical to those described above for use with any fluorometer. However, below we have outlined additional requirements that need to be followed in order to obtain meaningful results. As imaging is working on a relatively large illuminated surface, it is critical that the leaf or material of interest is held horizontal to the actinic and modulated lights in order to prevent heterogeneous illumination over different areas of the leaf. The material should also be held at the correct height relative to the light and camera in instruments that rely on in-built light calibrations (based on a distance) and do not provide a measurement of PAR at the measurement surface. Two elements are important for imaging chlorophyll fluorescence: the light source must provide even illumination over the entire surface of the imaged sample and the light source must be capable of providing a saturating pulse over the entire imaging area that is of sufficient intensity to close the majority of PSII centres and provide a representative Fm′. Saturating pulses of

2000 μmol m –2 s –1 are probably sufficient for dark-adapted Fm but may well be too low to provide a true Fm′ under high actinic illumination. Most commercial instruments provide between 4000 and 8000 μmol m –2 s –1 . If you are working with C4 plants, you may need higher saturating pulses. Before purchasing an instrument, it is worthwhile determining the instrument’s capabilities in terms of the intensity of the saturating pulse and how even the illumination is over the sampling area. Another difference between imaging systems and fibre optic systems is how Fo′ is determined, the value of which is required to calculate quenching parameters. Most fibre optic system incorporate LEDs that provide an FR pulse (see above). However, many commercial imaging systems rely on banks or panels of LEDs that provide the measuring beam and the actinic source, and they tend to be on one waveband and are therefore not capable of carrying out an FR pulse. Therefore, many imaging system calculate Fo′ using the mathematical algorithm developed by Oxborough and Baker (1997a) (Equation 1)

The use of this method to estimate Fo′ in situations where plants are stressed and may experience significant photoinhibition has been queried ( Maxwell and Johnson, 2000). However, this is not valid, as the only requirements for the calculation of Fo′ to be accurate are: (i) that PSII centres are open at the point of measuring Fo (ii) that there is no reversal of down-regulation between the measurements of Fo and Fm and (iii) that there is no reversal of photoinhibition between the measurements of Fm′ and Fm (for further information, see Lawson et al., 2002 Oxborough, 2004). It is been argued that the calculation of Fo′ is actually more accurate than the measured value, due to the difficulty in measuring Fo′ ( Lawson et al., 2002).

The wood from the trees: The use of timber in construction

Trees, and their derivative products, have been used by societies around the world for thousands of years. Contemporary construction of tall buildings from timber, in whole or in part, suggests a growing interest in the potential for building with wood at a scale not previously attainable. As wood is the only significant building material that is grown, we have a natural inclination that building in wood is good for the environment. But under what conditions is this really the case? The environmental benefits of using timber are not straightforward although it is a natural product, a large amount of energy is used to dry and process it. Much of this can come from the biomass of the tree itself, but that requires investment in plant, which is not always possible in an industry that is widely distributed among many small producers. And what should we build with wood? Are skyscrapers in timber a good use of this natural resource, or are there other aspects of civil and structural engineering, or large-scale infrastructure, that would be a better use of wood? Here, we consider a holistic picture ranging in scale from the science of the cell wall to the engineering and global policies that could maximise forestry and timber construction as a boon to both people and the planet.


In this essay I have tried to give the story of the development of a scientific infrastructure from a personal perspective. I hope that readers will understand that this all would have happened without me, but I did have a small part in the way it has turned out. In closing, I would like to reflect on a few things that I think have been important in helping me along.

The first is my collection of geniuses. This goes back to accidental mentors like Bob Loomis, to my graduate student days, to my time in Canberra, and to my association with the EOS team. But most of all, it has been my colleagues at Carnegie and Stanford—especially Olle Björkman and Hal Mooney.

An additional element has been the freedom provided in this environment. The Carnegie Institution has always provided a modest level of support that we could use as we chose, and has also helped to arrange similar support from other foundations. We could, and were encouraged to, take a long-term view in our research. I have also received generous funding from what I will call competitive grant programs. When I look back, I see that these were very important, but a lot of what I would characterize as the creative breakthroughs of my career came from the slow and steady support from Carnegie and the Mellon Foundation.

A remarkable exception is the NASA funding through the EOS team. This program had a long-range goal and long-range funding. The agency let us choose our team and develop our own approach. We could be bold in a way that could never be justified on a typical grant cycle. The similarity of this funding model to venture capital input for a start-up—the engine of innovation in the high-technology area—is obvious. Perhaps it should be used more often.

Watch the video: Xylem and Phloem - Transport in Plants. Biology. FuseSchool (June 2022).


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