PbHCT4 regulates growth through affecting chlorogenic acid (CGA) content in pear

Highlights

• The chlorogenic acid contents between dwarf and standard pear trees were different significantly.

• Low concentration of chlorogenic acid promoted the growth of pear, while high concentration of chlorogenic acid inhibited the growth.

• PbHCT4 gene changed plant height by regulating chlorogenic acid content and auxin transport.

Abstract

Due to its many advantages, dwarfing and dense planting is an important mode of modern fruit tree cultivation. Phenols have been reported to play various roles in the growth regulation of plants. Here, to verify the correlation between chlorogenic acid (CGA) and the growth of pear trees, the CGA content of standard and dwarf pear trees as well as the effect of exogenous CGA upon their growth, were investigated and analyzed. Our results showed that the CGA content of two dwarf-type varieties, ‘Zhongai 1′ (Z1) and ‘PY-9′ was significantly higher than that of two standard-type Pyrus betulaefolia (P1 and P2). Whereas a low concentration of CGA accelerated pear tree growth, and a high concentration of CGA could inhibit it. Then, through an expression pattern analysis of four CGA synthesis-related genes, PbHCT1–4, we found that the dwarf phenotype might be related to the expression levels of PbHCTs, especially to those of PbHCT2–4 in shoots. Of these, PbHCT4 was selected as a candidate gene and transformed into tobacco. When compared with control tobacco plants, the transgenic plants attained taller heights and had a greater CGA content, as well as increased expression levels of the auxin efflux carrier gene, NbPIN. Accordingly, we speculate the PbHCT4 gene promotes the synthesis of CGA, which influences plant growth by mediating auxin's transport in pear trees. These findings are helpful for advancing analysis of dwarf mechanism, which is critical for the breeding of new dwarf pear varieties.

Introduction

Compared with the traditional large-crown cultivation mode of fruit trees, the use of dwarfing and dense planting offers the advantages of early flowering (Foster et al., 2017), high yield and quality (Ou et al., 2019), labor-saving management (Kim et al., 1995), mechanized operation, and rapid variety renewal, making it an indispensable mode of modern fruit tree cultivation (Xiao et al., 2019). Dwarfing rootstocks or dwarf varieties are usually used for dwarfing and dense planting. For pear (Pyrus), many kinds of dwarfing rootstocks now exist, such as the quince series, OHF (Old Home x Farmingdale) series (Pyrus communis), Pyrodwarf (P. communis), Brossier (P. nivalis), Retuziere (P. communis), the S series (P. ussuriensis  ×  communis), PDR54 (P. ussuriensis  ×  communis), the Qingzhen series (P. xerophila Yü.), the Zhongai [(P. ussuriensis  × communis)  ×  spp.] series, and the K series (P. communis) (Cai et al., 2013). Yet, because of either grafting incompatibility or propagation difficulties of self-rooted rootstocks, these dwarfing rootstocks are seldom used for pear production in China. The cultivation of dwarf varieties is another way to solve this problem, whose breeding depends critically on dwarf mechanism analysis.

Concerning the dwarf mechanism, it typically results from reduced cell division or elongation, and these processes are generally regulated by phytohormones, including auxin (indole-3-acetic acid, IAA), brassinosteroids (BR), gibberellic acid (GA), and abscisic acid (ABA) (Michalczuk, 2002; Ma et al., 2016; Foster et al., 2017; Zhang et al., 2018; Liu et al., 2021). With respect to pear trees, IAA, BR and ABA have been reported to participate in their dwarf mechanism. Among these crucial plant hormones, IAA is considered among the most pivotal for enabling the dwarf mechanism (Zheng et al., 2019). PcPIN-L expression is significantly lower in dwarf-type pears, which is caused by the CT repeat deletion in its promoter. Through PcPIN-L overexpression in tobacco plants, it was verified that the low expression of PcPIN-L limits the polar auxin transport, thereby generating the dwarf phenotype (Zheng et al., 2019). IAA-induced miR171f negatively regulates the IAA signal cascade through GRAS pathway to maintain apical dominance, which uncovered a role for the miR171-SCL pathway in dwarfing Zhongai 3 (Jiang et al., 2018). Recently, Xiao et al. (2019) discovered a gene encoding an arabinogalactan protein (AGP) 7-like with high expression in dwarf pear plants. Transgenic pear lines overexpressing PcAGP7–1 exhibited obvious dwarf phenotypes, whereas RNAi interference (RNAi) pear lines were taller than the controls. PcAGP7–1 overexpression reduces brassinolide (BL) content, which inhibits BR signaling via a negative feedback loop, resulting in further dwarfing (Zheng et al., 2022). Further, it was found that ABA induced 601T, a strong mutant of dwarf pear 601D, to restore the dwarf phenotype; hence, the dwarf phenotype of 601D arises form an excessive accumulation of ABA (Liu et al., 2021).

In addition, the various phenols in some plants can play a role in their growth regulation. For example, in apple trees, vigorous and semi-vigorous rootstocks had higher phenolic contents than the dwarf rootstocks (Lockard et al., 1982; Yildirim et al., 2016). But some other researches reported that the levels of many phenols in the bark are more than adequate to induce apple tree growth inhibition, while benzoic acid, found only in their roots, also proved to be a potent growth inhibitor (Lockard et al., 1981). Researchers also proposed a dwarfing mechanism for apples, in which the phenols present in the bark of the rootstock play a key role in inhibiting the growth of scions (Kviklys et al., 2014). And phloridzin content in roots might play a role of inhibiting growth in apple and be used in dwarf rootstocks selection (Palfitov, 2003). Meanwhile, the growth of Lemna Paucicostata and Arabidopsis thaliana were inhibited by phenols (Park et al., 2012; Xu et al., 2017). In pear, its branch length is positively correlated with the contents of some polyphenols, including CGA (chlorogenic acid), epicatechin, rutin, and quercetin (Ren et al., 2015). As a kind of typical polyphenolic compound, CGA applied exogenously to lettuce could promote its growth at lower concentration but suppress its growth at higher concentration, mainly by affecting cell division (Chen et al., 2017). Indeed, CGA has been reported to function as an auxin protector, one capable of inhibiting the activity of auxin oxidase to prevent auxin from being degraded, thus promoting the occurrence of adventitious roots in plants (Pilet, 1964; Stonier et al., 2010). Nevertheless, CGA shares a common metabolic pathway with lignin and is an important intermediate in lignin biosynthesis. When a high concentration of CGA is applied, the synthesis of caffeoyl-CoA is intensified, which augments lignin's accumulation and the inhibition of adventitious roots in soybean (Liu et al., 2016).

Although some researches indicate the CGA content can affect plant growth and development, no direct evidence of a relationship between CGA and the growth of pear trees has yet been reported in the literature. In this study, by carrying out a CGA content analysis of standard-type Pyrus betulaefolia (P1, P2) vis-à-vis dwarf-type pear trees (PY-9 and Zhongai 1 [Z1]), as well as examining the effect of exogenous CGA on their growth, the correlation between CGA content and growth of pear trees was verified. From an expression pattern analysis of CGA synthesis-related genes PbHCT1–4 in P1 and PY-9, PbHCT4 was selected as the candidate gene and its function in CGA synthesis and influence upon plant growth was verified in transgenic tobacco. These results are helpful for studying the effect of chlorogenic acid on pear growth and the regulatory role of PbHCT4 in pear tree growth, which is critical for the breeding of dwarf pear varieties.