Guangping WU, Weixin CHEN, Xiaolin PAN, Siyin XU, Chengxiang XU*

1. Life Sciences College of Zhaoqing University, Zhaoqing 526061, China; 2. School of Life Science, Guangzhou University, Guangzhou 526061, China

Abstract The formation mechanism of heartwood is a scientific problem which has not been fully revealed in wood science. Effective regulation of heartwood formation is a scientific problem that is rarely studied and has not yet achieved a breakthrough in the field of forest cultivation. It is very beneficial to regulate the chemical properties of wood and directional and efficient cultivation of trees by fully understanding the formation mechanism of heartwood. Based on the author’s research and practice, the recent research progress on the formation mechanism and regulation technology of heartwood is summarized, especially the research on the heartwood formation of Hongmu species.

Key words Heartwood, Hongmu species, Directional cultivation

1 Introduction

The transverse section of branches with larger diameters is mainly composed of sapwood and heartwood. Sapwood is the part near the bark with lighter color, relatively soft property, higher moisture content and living cells, which mainly transports, transfers and stores nutrients. Heartwood is the part near the pith with darker color, relatively hard property, lower moisture content, no living cells, and no conduction and storage functions. Trees whose heartwood color is significantly different from sapwood are called "apparent heartwood trees", otherwise "saphenous heartwood trees". Wood that darkens and resembles heartwood due to fungal infection, mechanical damage,

etc.

is often called "false heartwood". The narrow area between sapwood and heartwood is called the transition zone or intermediate wood ("transitional wood" in Japanese).The texture of normal heartwood is stable, not easy to be deformed, moth-eaten and decayed. It is the best and most valuable part of logs, and even very precious wood or medicinal material. Therefore, although heartwood has no physiological function, it has very important value and influence on harvest and utilization of wood. However, it usually takes quite a long time for heartwood to grow to a large enough size. Currently, the scientific mechanism of heartwood formation is not well understood worldwide, and reports are scattered, limited to a few species. The heartwoods of

Robinia

pseudoacacia

,

Juglans

nigra

and

Cryptomeria

japonica

have been dedicated much more efforts in foreign countries; followed by

Pinus

spp.,

Fagus

sylvatica

and

Eucalyptus

robusta

. But even for the first three species, there have been very limited heartwood research literatures so far. The formation mechanism of heartwood is a scientific problem that has not been fully revealed in wood science, and the effective regulation of heartwood formation is a scientific problem that is rarely studied and has not yet achieved a breakthrough in the field of forest cultivation. It is very beneficial to regulate chemical properties of wood by fully understanding the formation mechanism of heartwood.

2 Hongmu: heartwood of a particular species of tree

Wet heartwood disease in

Populus

spp. has been studied greatly in China, so as to prevent and control the disease. In recent years, the technology promoting heartwood formation of Hongmu species, especially

Dalbergia

odorifera

, has attracted the attention of Chinese scientists and technicians, but the research on heartwood formation mechanism is still in the initial stage. Promoting "fragrance bearing" of precious tree species, such as

Santalum

album

,

Aquilaria

sinensis

and

Aquilaria

agallocha

, has attracted much attention in the industry. However, the "fragrance" formed by these species is actually the oil formed by resin secreted and fungus. It is related to but varies greatly with heartwood, and it is not heartwood in the scientific sense.

Hongmu is a conventional general term for commercial materials of a specific tree species, and specifically refers to the heartwood of this kind of wood. Hongmu has excellent properties, dark stripes and natural texture, and is loved by the public. It is a high-quality commodity material formed naturally over a long period of time, and a world-class high-end and precious wood with the basic characteristics of "hard, heavy, fine and red/black", mainly used for making high-grade furniture, carvings, musical instruments and arts and crafts (Fig.1). Chinese Hongmu product has a style of its own, and is well known in the world for its rarity of raw materials, unique practicality, artistry and value-added property.

Source: The first picture is shot by Xu Chengxiang, and the remaining pictures are derived from the Internet.

Hongmu and its forest resources have become an important national strategic resource and the focus of international timber market competition, and are precious wood to meet people’s demand for high-grade wood products. Currently, the international Hongmu resources are increasingly exhausted, and each producing country strictly controls trade and export. In

Convention

on

International

Trade

in

Endangered

Species

of

Wild

Fauna

and

Flora

(CITES) implemented in 2013, 7 species of controlled Hongmu species are added. More than 98% of China’s Hongmu depends on imports, and only one species of Hongmu,

D.

odorifera

, is distributed in China. Vigorously cultivating Hongmu forest resources is one of the major development strategies of tropical and sub-tropical forestry in China.According to the

National

Standard

of

Chinese

Hongmu

(GB/T 18107-2000), the timber of Hongmu can be divided into 8 categories, belonging to 33 species, 5 genera, 3 families. In the latest revision of

National

Standard

of

Chinese

Hongmu

(GB/T18107-2017), there are 29 species of Hongmu species, two species of red sandalwood (synonyms), one species of yellow sandalwood and ebony are cancelled and merged into one species, and Indian ironwodd is revised as

Cassia

L. Thus, there are six species of Hongmu in

Pterocarpus

, including

P.

santalinus

,

P.

dalbergioides

,

P.

erinaceus

,

P.

indicus

,

P.

macrocarpus

and

P.

marsupium

. Anyway,

P.

santalinus

is the most rare and precious tree species of Hongmu.

P.

santalinus

is mainly produced in India (Andhra Pradesh) and Myanmar. These countries and regions not only have strict restrictions on the export of

P.

santalinus

wood, but also have strict control over its seed trade. There is no natural distribution of

P.

santalinus

in China. In the 1950s, along with the introduction and cultivation of

Hevea

brasiliensis

, Hongmu species such as

P.

santalinus

were introduced to Hainan Island of China through folk channels and cultivated in small quantities. The practice of more than half a century shows that

P.

santalinus

grows well and is very suitable for cultivation in the south subtropical region of China. It is expected that

P.

santalinus

will be widely used in afforestation and landscape construction in south China, southern Yunnan and coastal areas of Fujian as more and more people know Hongmu species in China.Similar to other Hongmu species, the heartwood of

P.

santalinus

is formed late and has a long or even longer timber production cycle. It is generally believed that it takes about 20 years to form valuable heartwood, but

P.

macarocarpus

, which belongs to the same species, has a much earlier formation time (5 years) and a shorter timber production cycle (20 years). In the study on efficient propagation of

P.

santalinus

seedlings, professor Xu Chengxiang performed single bud bark grafting with cuttings at resting period using 1-year-old plants of

P.

macrocarpus

as the stock and 1-year-old robust branches of

P.

santalinus

as the scion, and received good survival rate and growth. Therefore, a new technique suitable for efficient propagation of

P.

santalinus

was explored, which significantly promoted the development of

P.

santalinus

heartwood. In 2020, the technique was authorized by the National Invention Patent. It has greatly enriched the cultivation theory and technology of Hongmu species, with outstanding practical value and scientific significance, provides very good materials for the study of heartwood formation mechanism, and is beneficial to the scientific understanding of formation mechanism of Hongmu heartwood and the scientific formulation of regulation technology.

3 Formation mechanism of heartwood

Heartwood is featured by stable property, beautiful texture, no deformation, moth or decay, more suitable for fine processing, and much value higher than sapwood, but the timber production cycle is very long. Therefore, in the production practice of forestry and medicinal materials, people try to take a variety of technical measures to promote the formation of heartwood, such as mechanical trauma, drought stress, injection of growth regulation substances,

etc.

. But in addition to a few precious tree species with "fragrance bearing" and harvesting resin (not heartwood in the scientific sense) as the goal, such as

S.

album

,

A.

sinensis

, there has been little overall success so far, and only partially colored, discontinuous and uneven "alienated wood" can be obtained. The main reason is that the heartwood formation mechanism is not well understood, so there are no scientific, targeted and effective measures.

Early research papers on heartwood formation described that since the transformation of wood ray of heartwood and radial parenchyma cells of sapwood was related to the aging of these cells, it was believed that heartwood was formed in hardwood because of the formation of air system in closed vessels, and various changes of parenchyma cells stimulated by oxygen were related to heartwood formation. Tea polyphenols would accumulate to toxic degree in inner sapwood, and heartwood formation is related to parenchyma cell death. Some scholars also suggest that the essence of heartwood formation is the existence of sapwood fungi in cells, and heartwood formation is the result of the decrease of water content of medial sapwood.

It is generally accepted that heartwood formation is due to decreased activity of ray parenchyma cells, increased distance between cambium and ray parenchyma cells, and imbalance of hormone balance. During the formation of heartwood, high level of cell activity occurs in the inner side of sapwood and the transition zone from sapwood to heartwood, and the primary formation zone is derived from the decline and fall of parenchyma cells in the inner side of pith. Wood ray parenchyma cells and small parenchyma cells in the peripheral region of pith may be involved in the synthesis of heartwood. The cellular respiration rate of sapwood adjacent to heartwood is significantly increased, while the mitochondrial activity of wood ray parenchyma tissues in sapwood is generally low. The mass development of cell wall materials also causes rapid cellulose methylation in many organs such as conduits, and leads to accumulation of secondary walls in radial resin canal tissues of pine trees, or even to the direction of occluded cells.

Heartwood and sapwood are easy to be distinguished, mainly attributed to the accumulation of pigment. Dark heartwood is more resistant to corrosion, so the color of heartwood is a sign of the presence of preservative substances, from which biological stains can be extracted. The heartwood formation is often accompanied by the production of a large number of thylose (also produced in sapwood injury and disease infection). The thylose is the parenchyma cell near the vessel, which is cystic and protrudes into the vessel from the pit. In the early stage, cytoplasm and nucleus flow into the vessel, and resin, colloid, tannin and crystal fill in the vessel in the later stage, which causes partial or total occlusion of vessel molecules, thereby reducing or losing the conduction ability. The thylose can develop secondary walls and even differentiate into stone cells, reduce the permeability of vessels, and block oxygen and mycorrhizal invasion.

The heartwood formation of

Quercus

L.,

Robinia

L. and

Morus

L. is related to the development of a large number of thylose. The occurrence of thylose in vessel and ray parenchyma cells is closely related to histidine. Histidine promotes secretion of polyphenol substances, which are toxic to protoplasm and will induce heartwood formation at lethal concentrations. Hormones can initiate conversion from sapwood to heartwood: ethylene in monterey pine leads to the production of polyphenols in heartwood, and abscisic acid is present in many types of sapwood. The weight, hardness, corrosion resistance and durability of heartwood are significantly increased by the formation of thylose and accumulation of related substances.

The formation of heartwood is a little weird. Trees seem to do well in the absence of heartwood, but most trees produce heartwood. Trees of the same age with larger diameters have a larger proportion of heartwood. Why is heartwood formed? What is the mechanism? In summary, the academic circles tend to accept that the formation of heartwood is a phase change, a development process, and a mature phenomenon. Transformation of sapwood and heartwood is an indicator of tree growth process, which essentially is a kind of regulatory mechanism promoting internal redundant sapwood into inert material to facilitate the centripetal transfer of active substances, and better meet the requirements of living cells of sapwood for mineral nutrients such as phosphorus and carbohydrates; as a result, the sapwood quantity and function is kept at the optimal level, and trees have strong enough activity, which avoid the fixation of internal sapwood for large amount of nutrition and greatly enhance the support function of the trunk. Therefore, the persistent death of many cells in the wood parenchyma tissues and ray tissues near the pith appears to be due to the accumulation of metabolic waste, which is an inevitable consequence of heartwood formation, but may also be due to the death of many cells in the secretory tissues.

The survival rate of

P.

santalinus

grafted and bred by professor Xu Chengxiang research group at Zhaoqing University in Guangdong reached more than 80% after 5 months, and the newly formed

P.

santalinus

plants grew well. The research team also found that the diameter of heartwood of each part within 60 cm above the graft union after 28 months increased by 64.8% and 29.6%, respectively, compared with that of non-grafted

P.

macrocarpus

and

P.

santalinus

trees of the same age, which had outstanding practical value and scientific significance for the breeding, cultivation and improvement of

Pterocarpus

trees. This paper was published in

Agricultural

Biotechnology

. There have been no similar reports at home or abroad, and the mechanism is still unclear.

4 Research themes and dynamics of heartwood

4.1 Dynamics at abroad

Since the 1990s, studies on the formation mechanism of heartwood have attracted more attention, but there are still very few literatures. From 1990 to 2018, 43 papers on heartwood were searched from journals published by world-renowned Elsevier and Springer-Verlag, with an annual publication volume of 1.483. Germany, the United States and Japan are the countries with a lot of research in this field, followed by France, Finland and Canada, while the United Kingdom, Australia, Switzerland, Austria, Spain, Portugal, Slovakia and Slovenia have also published research papers, and Mainland China and Taiwan each have one research paper.Among these heartwood literatures,

R.

pseudoacacia

,

J.

nigra

,

C.

japonica

are the most studied tree species, and the number of papers per tree species is ≥5. There are six species of trees with 2-3 papers, including

Larix

kaempferi

,

Fagus

sylvatica

,

Eucalyptus

robusta

,

P.

sylvestris

,

P.

pinaster

and

Thuja

plicata

. There are 10 species of trees with only one paper, including

Beilschmiedia

tawa

,

Juglans

nigra

,

Pseudotsuga

menziesii

,

Picea

sitchensis

,

Tectona

grandis

,

Erythrophleum

fordii

,

Taiwania

cryptomerioides

and

Acer

saccharum

. Overall, the researches are very scattered.In the above studies, the growth regularity of heartwood, especially the distribution of heartwood of different tree species on the longitudinal and transverse sections of the trunk, is still one of the research topics, such as formation prediction of

F.

sylvatica

heartwood, parameter prediction of

T.

grandis

heartwood in plantation, simulation of formation process of

E.

fordii

heartwood, heartwood, sapwood, bark, ring width and taper of

P.

menziesii

, biomechanical and hydraulic functions of irregular heartwood formation of

P.

pinaster

, relationship between irregular heartwood formation and centrifugal growth, radial growth and stem elongation growth, effect of heartwood formation on discolored wood proportion of

A.

saccharum

, formation of red heartwood of

F.

sylvatica

caused by bark stripping. Meantime, the formation mechanism of heartwood has become the most hot topic, such as chemical and biochemical metabolism in heartwood formation, cytological observation of water loss and reimmersion in tracheids, organelles and fluorescence of parenchyma cells of sapwood and heartwood, relationship of element content and pH value with change of heartwood color, participation of peroxidase in brown coloring of

J.

nigra

heartwood, association of polyphenols with the formation of

P.

menziesii

heartwood, relationship between oxide pentose phosphate pathway and pyridine nucleotide and heartwood formation of

R.

pseudoacacia

, relationships between activities of sucrose phosphate synthase, sucrose synthase, neutral invertase and protein content of

R.

pseudoacacia

with wood yield and heartwood formation in cambium. Isolation and identification of pharmaceutical components from heartwood has always been an important research topic, such as distribution of active ray parenchyma cells and main bioactive substances in

T.

cryptomerioides

heartwood, isolation and structural elucidation of norlignan polymers from the heartwood of

C.

japonica

, radial distribution of monomeric, dimeric and trimeric norlignans and their polymerization in

C.

japonica

heartwood.Molecular biology, especially various omics, has been introduced into the study of heartwood formation, such as high expression of homeobox gene in

J.

nigra

heartwood, cross-species transcriptional profiling analysis of

J.

nigra

heartwood formation, transcriptional localization of dirigent protein and 18S RNA of

T.

plicata

with heartwood formation, application of TOF-SIMS technology in heartwood formation of

C.

japonica

, expression of genes encoding chalcone synthase, flavanone 3-hydroxylase and dihydroflavanol 4-reductase related to flavanols during heartwood formation of

J.

nigra

, high expression of a KNAT3 homeobox gene derived from

J.

nigra

during heartwood formation, genetic variation in heartwood properties and growth traits of

E.

bosistoana

, transcriptomic analysis of juvenile wood formation during the growing season in

P.

canariensis

, developmental changes of

P.

sylvestris

transcriptome during heartwood formation.

4.2 Dynamics at home

Up to now, 136 literatures containing "heartwood" have been published in China. The earliest paper was published in 1958, and there were 16 papers published before 1995. In the following 10 years, the number of papers published increased significantly, reaching 2.6 papers per year. After 2006, the annual average number of papers published is more than 6.2 per year (Fig.2). These papers have been published in 65 journals (including degree-granting units), covering 43 species of tree species so far.

Populus

spp. are the most studied (25 papers), followed by

L.

kaempferi

,

D.

odorifera

, and fir wood, each with 14 papers. There are 6-8 research papers on heartwood formation of cypress, pine, locust and sandalwood.

Fig.2 The number of Chinese papers on heartwood research in different periods (based on CNKI database resources)

The top five research topics are chemical composition and detection of heartwood, bacteriological and anticorrosive effect of heartwood, heartwood rot, poplar wet heartwood, heartwood proportion and wood density, followed by the research on technologies promoting heartwood formation. Before 2000, heartwood diseases were mainly studied. Since 2000, the topics such as heartwood ratio and characteristics of heartwood species, chemical analysis of heartwood components, antibacterial effect and application of heartwood extract have been greatly published.

5 Conclusions

Based on the author’s research and practice, the recent research progress on the formation mechanism and regulation technology of heartwood is summarized, especially the research on the heartwood formation of Hongmu species. In the last 10 years, attention has been paid to the techniques promoting the heartwood formation of precious tree species, especially

S.

album

and

D.

odorifera

, but there has to be innovation in technology.