Elsevier

Plant Science

Volume 298, September 2020, 110544
Plant Science

Suppression of a hexokinase gene, SlHXK1, leads to accelerated leaf senescence and stunted plant growth in tomato

https://doi.org/10.1016/j.plantsci.2020.110544Get rights and content

Highlights

  • Suppression of SlHXK1 lead to accelerated leaf senescence and stunted plant growth.

  • SlHXK1-RNAi lines generates shorter leaves and petiole.

  • Down-regulation of SlHXK1 causes more starch to accumulate in tomato leaves.

Abstract

Sugars are the key regulatory molecules that impact diverse biological processes in plants. Hexokinase, the key rate-limiting enzyme in hexose metabolism, takes part in the first step of glycolytic pathway. Acting as a sensor that mediates sugar regulation, hexokinase has been proved to play significant roles in regulating plant growth and development. Here, we isolated a hexokinase gene SlHXK1 from tomato. Its transcript levels were higher in flowers and leaves than in other organs and decreased during leaf and petiole development. SlHXK1-RNAi lines displayed advanced leaf senescence and stunted plant growth. Physiological features including plant height, leaf length, thickness and size, the contents of chlorophyll, starch and MDA, and hexokinase activity were dramatically altered in SlHXK1-RNAi plants. Dark-induced leaf senescence were advanced and the transcripts of senescence-related genes after darkness treatment were markedly increased in SlHXK1-RNAi plants. RNA-seq and qRT-PCR analyses showed that the transcripts of genes related to plant hormones, photosynthesis, chloroplast development, chlorophyll synthesis and metabolism, cellular process, starch and sucrose metabolism, and senescence were significantly altered in SlHXK1-RNAi plants. Taken together, our data demonstrate that SlHXK1 is a significant gene involved in leaf senescence and plant growth and development in tomato through affecting starch turnover.

Introduction

Leaves are usually composed of epidermis, mesophyll and veins, which initiate from the shoot apical meristem (SAM) [1,2]. The initiation of plant leaves begin from the leaf primordia and develop into photosynthetic organs of a specific size and shape by cell division, differentiation and expansion [3,4]. Finally, leaves enter the aging phase [5]. Leaf senescence is essential for plant survival and fitness because it can effectively relocate the accumulated nutrients in the growth stage to other developing parts, such as the seeds, fruits and so on [6,7]. Leaf senescence is a genetically controlled process that is not only affected by environmental factors, but also regulated by many genes [4,8]. Leaf senescence leads to large-scale but orderly changes in plant physiology, biochemistry, and molecular events, such as chlorophyll degradation, macromolecular hydrolysis and reactivation, a decline in photosynthetic activity and programmed cell death [4,9]. It involves tight control of multi-layer regulation, such as chromatin and transcription levels, as well as translation and post-translational regulation levels [9]. Loss of chlorophyll is an early manifestation of leaf senescence. Originally, the mesophyll cells begin to age, then other types of cells follow. Further, leaf senescence exhibits a discontinuous pattern of local cell death that finally spreads throughout the leaf [10].

In recent years, many studies have reported that numerous phytohormones including abscisic acid (ABA), gibberellins (GAs), ethylene and jasmonic acid (JA) and multiple transcription factors, such as NAC, MYB and WRKY were reported to participate in the regulation of leaf senescence. SlNAP2, an NAC transcription factor from tomato, that controls both fruit yield and leaf senescence [11]. In tomato, overexpression of NOR, which is one of the best characterized NACs, leads to accelerated senescence, while senescence is delayed in the nor mutant [12]. Zhang X et al.(2011) reported that AtMYBL is a R-R-type MYB-like transcription factor participated in the regulation of leaf senescence [13]. Overexpression of the AtWRKY22 gene in Arabidopsis thaliana accelerates dark-induced leaf senescence phenotype, but the AtWRKY22-suppressed plants displays the delayed senescence leaves [14]. In addition to the significantly roles of transcription factors in leaf senescence, some enzyme genes also are reported to play essential roles in this process. For instance, RPK1, a membrane-bound receptor protein kinase, regulates age-dependent and abscisic acid (ABA) induced leaf senescence in Arabidopsis thaliana, a phenotype of prk1 mutants exhibiting aging delay in ABA-induced senescence [15]. It has been reported that SAG101 encodes an acyl hydrolase gene involved in the regulation of Arabidopsis thaliana leaf senescence, antisense inhibition of this gene delays leaf senescence, and chemically induced ectopic overexpression accelerates leaf senescence [16]. In addition, AtSARK, a homolog of the soybean bispecific kinase GmSARK in Arabidopsis thaliana, is regulated by the synergistic action of ethylene and auxin to regulate leaf senescence. Overexpression of AtSARK leads to accelerated senescence and abnormal floral organ development, while T-DNA insertion mutant of AtSARK exhibits a senescent delayed phenotype [17].

As a central compound in nature, sugar plays a key role in the process of gene expression, metabolism, and cell cycle and development in eukaryotes and prokaryotes [[18], [19], [20]]. In higher plants, sugars play significant roles in controlling growth and development at different stages and periods of their whole life cycle, such as the process of plant germination, growth, flowering and senescence [21,22]. The hexokinase which is encoded by hexokinase genes (HXKs) is the key rate-limiting enzyme of glycolysis metabolism and plant respiration. It is found to function as the glycolytic enzyme to catalyze the transition of hexoses into hexose 6-phosphates in the first step of glycolysis pathway for sugar metabolism and accumulation. As the glucose sensor, it has been shown to play key roles in sugar signal transduction and plant growth and development [23,24].

Plant HXK is encoded by a medium family of approximately 4–10 genes [25]. Overexpression of Arabidopsis thaliana AtHXK1 gene inhibited the expression of photosynthetic genes and accelerated senescence. Studies on AtHXK1 transgenic plants and gin2 (glucose-insensitive 2) mutants show that HXK1 is a true glucose sensor [24,26]. Under light limiting conditions, the glucose sensor HXK1 carry out a basic role during post-germinative growth. Hexokinase (HXK) is also involved in signaling pathways, for instance AtHXK2 is induced under osmotic, salt stress or cold [27]. In addition to glucose-induced signaling, AtHXK1 mainly functions as a glycolytic enzyme in Arabidopsis thaliana seedlings [28]. Furthermore, Arabidopsis thaliana hexokinase1 (AtHXK1) participates in cell death mediated by the accumulation of myo-inositol and reduces transpiration and controls stomatal aperture [29,30]. Repression of hexokinase gene OsHXK10 mediated by RNAi approach in rice results in the decrease of pollen germination and non-dehiscent anther, showing that OsHXK10 gene plays a key role in pollen germination and anther dehiscence [31]. Besides, the gin2-1 mutant (glucose‐insensitive mutant) is complemented by expression of either OsHXK5 or OsHXK6 and overexpressing OsHXK5 or OsHXK6 displays hypersensitive to glucose and repression of plant growth in rice, indicating that OsHXK5 and OsHXK6 can be used as sugar sensors [32]. In tobacco, NtHXK2 is localized to the chloroplast matrix and may be involved in glucose phosphorylation derived from starch breakdown [33]. Moreover, silencing of NtHXK1 led to severely damaged phenotypes in leaves and chloroplasts, and NtHXK1 can be able complement the Arabidopsis thaliana mutant gin2‐1 (glucose‐insensitive) [34].

As the final stage of leaf development, the leaves undergo a large number of genetic changes throughout the aging process, and regulating the expression of aging-related genes is a powerful strategy to control the aging of agronomic purposes [[35], [36], [37]]. Nowadays, tomato has become a model crop commonly used in investigation of plant growth and development. Four HXK isoforms have been identified in tomato [38]. In the present study, we isolated a hexokinase gene, SlHXK1 (Solyc03g121070.3) from tomato (Solanum lycopersicum, Mill. cv. Ailsa Craig, AC++). RNAi-mediated silencing of the SlHXK1 gene was performed to study the exact function of the SlHXK1 gene in tomato, and the SlHXK1-RNAi seedlings exhibited stunted growth and accelerated leaf senescence compared with the wild type (WT) under normal growing conditions. These phenotypes were further confirmed by morphological, statistic, biochemical, ultrastructure and molecular analysis in SlHXK1-RNAi lines. The results of our research indicate that SlHXK1 plays an important role in tomato seedling development and leaf senescence. This study strengthens our knowledge about the key roles of SlHXK1 in a variety of biological processes during tomato development.

Section snippets

Plant materials and growth conditions

In our experiments, the tomato (Solanum lycopersicum, Mill. cv. Ailsa Craig, AC++), was used as the WT. All tomato plants were germinated on MS medium and grown in the greenhouse under the following conditions: 25 °C for day (16 h) and 18 °C for night (8 h), 250 μmol m−2 s-1 light intensity, and 80 % humidity. The samples of roots, stems, flowers, leaves, petioles, inflorescence, sepals and fruits (GF and RF) were collected from wild-type tomato plants for SlHXK1 organ-specific expression

Sequence and expression analysis of SlHXK1

Here, a tomato hexokinase gene was isolated and named SlHXK1 (Solyc03g121070.3). Sequence analysis exhibited that SlHXK1 contains a 1497 bp ORF (open reading frame) encoding a protein with 498 amino acid and an estimated molecular mass of 54.038 kDa (pI 5.91). Multiple alignment results showed that SlHXK1 harbors conserved regions, including two phosphate sites (I and II), two connect sites (I and II), one sugar binding site, one conserved α-helix site and one adenosine binding site (Fig. 1A).

Discussion

So far, studies on HXK isoform characterization in different plant species have been performed. Hexokinase is known to be a sugar sensor that mediates both the sugar signaling and the catabolic activity of hexose phospholiration [56]. For instance, inhibited expression of OsHXK10, some flower anthers can not open (flowering) and release their pollen, resulting in reduced pollen sprouting, significantly increased the proportion of empty seeds, indicating that OsHXK10 plays an important role in

Author contribution statement

Zongli Hu and Qiaoli Xie designed and managed the research work and improved the manuscript. Jing Li, Guoping Chen, Jianling Zhang, Hui Shen, Jing Kang, Panpan Feng performed the experiments. Jing Li wrote the manuscript.

Declaration of Competing Interest

All authors have read and approved this version of the article, and due care has been taken to ensure the integrity of this work. The authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by Graduate Scientific Research and Innovation Foundation of Chongqing, China (CYS19012), National Natural Science Foundation of China (no.31872121, 31801870) and Natural Science Foundation of Chongqing of China (cstc2019jcyj-msxmX0361).

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