Senescence is delayed when ramie (Boehmeria nivea L.) is transformed with the isopentyl transferase (ipt) gene under control of the SAG12 promoter

Ramie is an economically important industrial fiber crop widely planted in China, India, and other Southeast Asian and Pacific Rim countries. It plays an important role in China's economy, where ramie farming, industry, and trade provide livelihood support to about five million people. However, poor fiber production resulting from leaf senescence and leaf abscission is a significant problem. In this study, we report the successful production of transgenic ramie plants which delayed leaf senescence and enhanced biomass. Transgenic ramie plants were obtained via transformation with the Agrobacterium tumefaciens strain harboring the binary vector pSG529 containing the isopentyl transferase (ipt) gene under control of the SAG12 promoter (PSAG 12‐ipt construct). Agrobacterium tumefaciens strain EHA105 was used for the midrib explant transformation. The transformation frequency was 28.29%. Southern blot confirmed the integration of 1–4 copies of the NPTII gene into the ramie genome in the tested lines. At the fiber maturation stage, the transgenic plants had higher photosynthesis rates, chlorophyll content (SPAD values), and stronger resistance to exogenous ethylene compared with wild‐type plants.

Ramie is an economically important industrial fiber crop widely planted in China, India, and other Southeast Asian and Pacific Rim countries. It plays an important role in China's economy, where ramie farming, industry, and trade provide livelihood support to about five million people. However, poor fiber production resulting from leaf senescence and leaf abscission is a significant problem. In this study, we report the successful production of transgenic ramie plants which delayed leaf senescence and enhanced biomass. Transgenic ramie plants were obtained via transformation with the Agrobacterium tumefaciens strain harboring the binary vector pSG529 containing the isopentyl transferase (ipt) gene under control of the SAG12 promoter (P SAG12 -ipt construct). Agrobacterium tumefaciens strain EHA105 was used for the midrib explant transformation. The transformation frequency was 28.29%. Southern blot confirmed the integration of 1-4 copies of the NPTII gene into the ramie genome in the tested lines. At the fiber maturation stage, the transgenic plants had higher photosynthesis rates, chlorophyll content (SPAD values), and stronger resistance to exogenous ethylene compared with wild-type plants.
Ramie (Boehmeria nivea L. Gaud) of the Urticaceae [1] is a perennial bast fiber crop that originated in China [2]. Therefore, it is known as 'China grass' in many western countries [1]. Ramie is grown on about 80 000 ha, with annual fiber production of 150 000t in 2012 (FAOSTAT, http://faostat3.fao.org). It has vigorous vegetative growth and can be harvested three times a year in the Yangtze River Basin [1] and up to six times a year in well-watered environments in the Philippines, which means that the ramie vegetative fiber yield is very high [3].
Poor fiber production resulting from leaf senescence (Fig. 1A) and leaf abscission (Fig. 1B) at the fiber maturation stage is a significant problem. Senescence is an orderly loss of normal cell functions under the control of the nucleus [4]. Leaf senescence is an important feature of the later stage of development and the age-dependent deterioration process, and leads to death [5]. Leaf senescence is illustrated clearly by dramatic color changes [6]. Green leaves on perennial plants including ramie turn yellow and orange before they eventually brown, die, and are discarded from the plant [6]. Leaves are specialized photosynthetic organs and the plant invests considerable energy and nutrients in leaf production [7]. During leaf senescence, the earliest and most drastic change in plant cellular structures is the breakdown of chloroplasts, which contain the photosynthetic machinery of the cell and carry out major biosynthesis [8]. Leaf senescence is characterized by a decline in chlorophyll content [9]. Chlorophyll Abbreviations Cks, cytokinins; IPT, isopentenyl transferase; PCD, programmed cell death; SAG, senescence-associated gene; WT, wild-type.
proteins and nucleic acids are degraded during senescence, resulting in a sharp decrease in leaf photosynthetic activity [10]. Leaf senescence can therefore substantially limit crop biomass accumulation. This complex process involves a sequence of changes in cellular physiology, biochemistry, and gene expression [11]. Senescence is a gradual process and therefore difficult to quantify [9]. Furthermore, leaf senescence can be induced by various internal and external environmental factors, such as light and a variety of plant hormones [5,12,13]. Many studies on senescence have been carried out to better understand the leaf senescence processes in tobacco [14], Arabidopsis [11,15], cotton [16], wheat [17], rice [18], and petunia [19]. Several investigations have demonstrated that leaf senescence can be induced or suppressed by various phytohormones [20]. Methyl jasmonate, salicylic acid, ethylene, abscisic acid, low nutrient supply, low light conditions, stress, and pathogen infection are all thought to trigger or enhance senescence, whereas cytokinins (Cks), gibberellins, and auxins have been reported to delay senescence [9,11,21,22]. Cks are the strongest senescence-delaying hormones [20,21]. Increased Ck production can delay leaf senescence, whereas reduced endogenous Ck levels can result in premature senescence [21].
The enzyme that catalyzes the rate-limiting step of the Ck biosynthesis pathway [23] in Agrobacterium tumefaciens is isopentenyltransferase (IPT) [24]. In an earlier study, leaf Ck concentrations were elevated and leaf senescence was delayed in transgenic plants because of overexpression of the ipt gene, but the high Ck levels were largely detrimental to growth and fertility [23]. When the ipt gene was expressed under the control of a senescence-inducible promoter (the SAG12 promoter), elevated Ck levels were localized within senescing tissues or organs and senescence was delayed without the induction of additional phenotypes associated with systemically high hormone levels [25]. This approach was later successfully applied to delay the senescence process in tobacco [26], tomato [27], lettuce [23], broccoli [28], and petunia flowers [24]. In addition to retardation of leaf senescence, some of these transgenic plants delayed flowering [23,24], increased resistance to ethylene [24,29] and drought [30,31], and enhanced root growth [30,32].
Previously, we developed an efficient regeneration and transformation protocol for ramie cultivar improvement by Agrobacterium-mediated gene transformation using leaf midribs as explants [2]. In the present study, we transformed the P SAG12 -ipt gene into the ramie cultivar 'Huazhu No. 5', which resulted in delayed leaf senescence and increased biomass compared with the wild-type.

Plant materials
'Huazhu No. 5' was used as the plant material in this study. It was prepared as previously described [2].

Agrobacterium-mediated transformation
The A. tumefaciens strain EHA105, harboring the binary plasmid pSG529, was donated by Professor Xianlong Zhang (Huazhong Agricultural University, China). Agrobacterium-mediated transformation was carried out as previously described [2].

Leaf senescence experiment
The stem diameter and height of randomly selected transgenic and WT plants were measured at the beginning (control) and end (treatment) of the fiber maturation stage. Six measurements were performed for chlorophyll content using a SPAD-502 plus (Konica Minolta, Tokyo, Japan) on corresponding functional leaves, and the average was taken. The photosynthesis rate was determined as previously described [33].

Measurement of biomass traits
The biomass traits of randomly selected transgenic and WT plants were measured at the beginning (control) and end (treatment) of the fiber maturation stage. The biomass traits included fresh shoot weight, fresh stem weight, fresh bast weight, and dry bast weight.

Data collection and statistical analysis
The biomass traits of randomly selected transgenic (lines 19 and 20) and WT plants were measured at the beginning (control) and end (treatment) of the fiber maturation stage.
Each treatment consisted of nine replicate pots (three WT plants and three plants of each transgenic line, one plant per pot). Each ethylene stress treatment consisted of nine replicate trays (three detached functional leaves from the WT and each transgenic line). All experiments were replicated three times and randomized. The regeneration frequency of Km-resistant shoots and the NPTII-positive frequency of the explants were calculated as previously described [2]. The data were recorded and analyzed statistically using SAS version 6.12 (SAS Institute, 1995, Cary, NC, USA). Significant differences among means were compared using Duncan's Multiple Range Test. All stress experiments were conducted in a greenhouse at 28 AE 2°C/ 23 AE 2°C (day/night) with a relative humidity of 50%-70% under a 16/8-h (light/dark) photoperiod and a photon flux density of 350 lmolÁm À2 Ás À1 .

Generation of transgenic ramie plants and transformation efficiency
The explant regeneration and transformation were shown in Fig. 2. Delaying senescence using the P SAG12 -ipt gene construct has also been reported in lettuce [23], Petunia flowers [24], Arabidopsis [34], gerbera [35], and creeping bentgrass [32]. Regeneration and transformation were performed using midrib explants from 40-day-old in vitro shoots, an Agrobacterium concentration at OD 600 of 0.6, 4-min immersion in the bacterial solution, an acetosyringone concentration of 50 mgÁL À1 in the cocultivation medium and a 2-day cocultivation period. The efficiencies of regeneration and transformation were 28.29% and 25.56%, respectively. The regeneration of transgenic plants was shown in Fig. 3. In this study, the regeneration frequency of Km-resistant shoots was higher than that previously reported [2].

Molecular analysis of the transgenic plants
The binary vector pSG529 harboring the Ck biosynthesis gene ipt, derived from A. tumefaciens, under the control of the SAG12 promoter from Arabidopsis was shown in Fig. 4A. Vegetative propagation is the main propagation method in practical ramie production and the materials here did not produce seeds; all plants used in this study were thus T0 plants [33]. Twentytwo putatively transgenic T0 ramie plants were regenerated and confirmed by PCR (Fig. 4B). Eleven of them (lines 1, 5, 7, 8, 9, 13, 15, 17, 19, 20, and 21) were randomly selected for RT-PCR (Fig. 4C) (Fig. 4D).

Analysis of chlorophyll content and photosynthesis in fresh leaves at the fiber maturation stage
The leaf relative chlorophyll content (SPAD values) and photosynthesis rates of randomly selected transgenic and WT plants were measured at the beginning (control) and end (treatment) of the fiber maturation stage. There were no differences in SPAD values or photosynthesis rates among the randomly selected transgenic and WT plants at the beginning of the fiber maturation stage. As expected, leaf senescence and leaf abscission at the fiber maturation stage had an adverse effect on SPAD values and photosynthesis rates (Fig. 5A,B; Treatment). However, the reductions in SPAD values and photosynthesis rates of the transgenic plants were less severe than those observed in the WT plants (Fig. 5A,B; Treatment). Leaf senescence is a type of programmed cell death (PCD) characterized by loss of chlorophyll, lipids, total protein, and RNA [36]. The potential for improvement of crop characteristics, particularly plant productivity and postharvest storage, has prompted extensive physiological, molecular, and genetic analyses of leaf senescence [37]. Endogenous factors, including leaf age and reproductive development, trigger senescence [36]. Leaf senescence at the fiber maturation stage is a biological process. In the WT, leaf senescence was initiated at the fiber maturation stage in naturally grown plants, which turned yellow (low SPAD values), whereas senescing leaves from the P SAG12 -ipt transgenic plants remained dark green (high SPAD values). As leaves are the main photosynthetic organs, the expression of ipt in leaves resulted in maintenance of chlorophyll content and higher photosynthesis rates compared with WT plants in this study.

Ethephon-induced leaf senescence analysis
Ethylene is known to promote the leaf senescence process [29]. To confirm that leaf senescence was delayed in ipt-overexpressing plants, an ethephon-induced leaf senescence test was conducted using the above two iptoverexpressing plants (T19 and T20) and WT plants.
Detached leaves of the transgenic and WT plants were incubated in the presence of 1200 ppm ethylene for 10 days in water (mock treatment) and photographed. After ethephon treatment for 3 days, WT leaves showed visible leaf yellowing, while the transgenic lines displayed delayed leaf senescence (Fig. 6). Ethephon treatment for 6 days had a clear effect on the WT plants, whereas transgenic lines 19 and 20 remained green (Fig. 6). Ethephon treatment for 10 days had a severe effect on the WT plants, which were completely wilted, whereas transgenic lines 19 and 20 showed only slight yellowing (Fig. 6). Therefore, our results indicated that ethephon-induced leaf senescence was delayed in the transgenic lines compared with WT plants. A reduction in sensitivity to ethylene has also been reported in P SAG12 -ipt transgenic petunia plants, where increased levels of Cks and ipt mRNA abundance were detected a few hours after ethylene treatment [24].

Analysis of biomass traits and phenotype in transgenic and WT plants
To confirm that biomass was increased in the ipt-overexpressing plants, the phenotype and biomass traits including height, stem diameter, fresh shoot weight, fresh stem weight, fresh bast weight, and dry bast  weight were measured. At the beginning of the fiber maturation stage, there were no significant differences among the WT and transgenic plants in phenotype (Fig. 7A), height, or stem diameter (Fig. 8, control). However, there were significant differences between the WT and transgenic plants in height, stem diameter, fresh weight, fresh stem weight, fresh bast weight, and dry bast weight (Fig. 8, treatment) at the end of the fiber maturation stage. At the fiber maturation stage, significant phenotypic differences (Fig. 7B) between transgenic lines 19 and 20 were observed. Our data showed that transformation of ramie with the P SAG12ipt gene delayed leaf senescence and enhanced biomass. Roses and stay-green lines have been obtained with similar features to those described in the present paper for ramie [29]. Because of the worldwide economic importance of ramie, the decreased senescence observed in transgenic P SAG12 -ipt ramie has commercial value. The transgenic plants described here provide a range of stay-green phenotypes to facilitate studies of the impact of leaf senescence manipulation on yield under field conditions.

Conclusions
In this study, the transformation efficiency was 25.56%, which was higher than that reported previously [2]. The transgenic plants had higher photosynthesis rates and SPAD values than wild-type plants at the fiber maturation stage. The transgenic plants also had enhanced resistance to exogenous ethylene compared with the wild-type plants at the fiber maturation stage. Overall, this study suggests that ipt gene overexpression delayed leaf senescence and enhanced biomass characteristics including height, stem diameter, fresh weight, fresh stem weight, fresh bast weight, and dry bast weight in transgenic plants compared with WT plants, and has the potential to improve ramie fiber yield.